Methods for stimulating the regeneration of retinal ganglion cells

Abstract

Compositions, nucleic acid molecules, and methods are described for inducing retinal regeneration and reprogramming of Muller glia (MG) into retinal ganglion cells in a subject. Developmental retinal ganglion cell (RGC) transcription factors Pou4f2 and Islet1 increase the Ascl1-induced neurogenic capacity of MG. The combination of Ascl1, Pou4f2, and Islet1 stimulates MG to generate bipolar cells and RGC-like neurons. Similarly, the transcription factor Onecut1, which is expressed in developing retinal cells but not in MG, induces MG to generate RCG-like cells. Additional transcription factors that can be used include Irx2, Irx5, Neurod2, Ebf1, and Tcf3. MG-derived RGCs can show action potentials in vivo and show chromatin profiles similar to developing RGCs.

Description

[Technical field]

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

[0002] Sequence Listing Reference The contents of the sequence listing XML file entitled "UW77_seq", created on March 30, 2023 and submitted electronically herewith, of 666 kb in size, are incorporated herein by reference in their entirety.

[0003] Recognition of government support 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 technology]

[0004] Neurodegenerative disorders of the eye result in 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), the primary glial cells in the vertebrate retina, can serve as a source of neurogenic precursors in regenerative species. However, in the mammalian retina, MG respond to retinal injury by undergoing an inflammatory response instead of a regenerative response.

[0005] There are many diseases that cause the loss of specific neuronal populations in the retina, resulting in blindness. Thus, there remains a need to stimulate regeneration in the human retina in order to develop new types of regenerative therapies for patients. Summary of the Invention

[0006] Described herein are compositions, nucleic acid molecules, and methods for inducing retinal regeneration and reprogramming of Muller glia (MG) into retinal ganglion cells in a subject. Examples provided herein show that developmental retinal ganglion cell (RGC) transcription factors Pou4f2 and Islet1 increase the Ascl1-induced neurogenic capacity of MG. The combination of Ascl1, Pou4f2, and Islet1 stimulates MG to generate bipolar cells and RGC-like neurons. Similarly, the transcription factor Onecut1, which is expressed in developing retinal cells but not in MG, induces MG to generate RCG-like cells. Additional transcription factors that can be used include Irx2, Irx5, Neurod2, Ebf1, and Tcf3. MG-derived RGCs can exhibit action potentials in vivo and show a chromatin profile similar to developing RGCs.

[0007] 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 comprises Pou4f2 and / or Islet1. In some embodiments, the RGC transcription factor comprises Irx2 and / or Neurod2.

[0008] In some embodiments, the nucleic acid sequence further comprises a nucleic acid sequence encoding a proneural basic helix-loop-helix (bHLH) transcription factor. In some embodiments, the proneural bHLH transcription factor and the RGC transcription factor are expressed as a fusion protein. Representative examples of proneural bHLH transcription factors include, but are not limited to, Ascl1, Atonal7 (also known as Math5), Atoh1 (also known as Math1), Neurogenin-2, and Neuronal Differentiation1 (Neurod1). In one embodiment, the nucleic acid molecule further comprises a nucleic acid sequence encoding Ascl1 and Atoh1. In one embodiment, the nucleic acid molecule further comprises a nucleic acid sequence encoding Ascl1 and Atoh7. In one embodiment, the nucleic acid molecule further comprises a nucleic acid sequence encoding Atoh1 and Atoh7. In one embodiment, the nucleic acid molecule further comprises a nucleic acid sequence encoding Ascl1, Atoh1, and Atoh7. In some embodiments, the nucleic acid molecule comprises a nucleic acid sequence encoding two, three, four, or all five of Ascl1, Atonal7, Atoh1, Neurogenin-2, and Neurod1.

[0009] 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.

[0010] In some embodiments, the nucleic acid sequence further comprises a promoter sequence operably linked to the nucleic acid sequence encoding the RGC transcription factor. In some embodiments, the promoter sequence is not naturally occurring operably linked to the nucleic acid sequence encoding the RGC transcription factor. In some embodiments, the promoter sequence is for a gene specifically expressed in glial cells, such as RLBP1 or GLAST. In some embodiments, the promoter sequence is the HES1, RLBP1, or GLAST promoter. In some embodiments, the promoter sequence precedes an effector gene CRE or tTA that drives transcription in an inducible manner. In one embodiment, the MG-specific promoter sequence is the Rbpl1 promoter sequence or a portion thereof. In other embodiments, the nucleic acid sequence comprises an IRES or 2A self-cleavage site located between the sequences encoding the transcription factor, e.g., in a multicistronic or polycistronic arrangement. In other embodiments, the nucleic acid sequence comprises an element that causes transcription of the gene in an inducible manner in response to the presence of CRE or tTA. These elements include, but are not limited to, loxP, lox2272, and tetracycline response element (TRE).

[0011] Also provided is a composition comprising the nucleic acid molecule described herein. In some embodiments, the nucleic acid molecule is mRNA. Such compositions can be optionally formulated for delivery 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) 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 injection or subretinal injection.

[0012] 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™, also known as (Pro) / romidepsin, Beleodaq™, also known as (Pro) / belinostat, Farydak™, also known as (Pro) / panobinostat, and Zolinza™, 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).

[0013] Also disclosed herein are methods for inducing retinal regeneration or stimulating retinal ganglion cell regeneration by administering to cells, such as 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 vivo or in vitro. 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 factors include Irx2 and / or Neurod2.

[0014] Also disclosed herein are methods for inducing retinal regeneration or stimulating regeneration of retinal ganglion cells in a subject. These methods include administering to the retina of the subject a nucleic acid molecule comprising a nucleic acid sequence encoding a developmental retinal ganglion cell (RGC) transcription factor. Exemplary RGCTFs 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.

[0015] 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 a first administration of a composition comprising a first nucleic acid molecule encoding a proneural bHLH transcription factor, and at a later time, a second administration of 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. 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.

[0016] In additional embodiments, the nucleic acid molecules and methods disclosed herein stimulate the production of functional RGCs from reprogrammed MG. In another embodiment, the number of functional RGCs from MG is increased. In another embodiment, the number of functional RGCs is increased by 40%. In another embodiment, the subject is treated for retinal disease, injury, or degeneration. In another embodiment, the subject is an adult. In another embodiment, the vector comprises the 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 viral (AAV) vector or a lentiviral vector. In additional embodiments, the 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, administering to the retina is an intravitreal injection or a subretinal injection.

[0017] Also provided herein is a method for inducing retinal regeneration, comprising administering to a subject a composition described herein. In some embodiments, the method is effective for increasing the number of Müller glia-derived RGCs, inducing Müller glia cells to enter the mitotic cell cycle, and / or generating new RGCs. In some embodiments of the method, the number of RGCs is increased by at least 40% relative to a baseline level or other reference amount representing an untreated retina. In some embodiments, the number of RGCs is increased by 10%, 20%, 25%, 50%, 100%, 150%, 200%, or more.

[0018] The subject in the method 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 has 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 syndrome.

[0019] Reprogramming MG and regeneration of retinal neurons is particularly important for developing therapeutics 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 known as central retinal artery occlusion (CRAO), in which blood flow through the central retinal artery is blocked or occluded, often leading to vision loss. CRAO is caused by thromboembolism, carotid atherosclerosis, giant cell arteritis, aneurysm, or arterial spasm. Current treatment paradigms for many of these types of retinal degenerative diseases, especially for CRAO, have shown little or no definitive improvement in outcome. Another indication could be paracentral acute mid-maculopathy from vein occlusion (CRVO) or other causes leading to inner retinal thinning and vision loss. [Brief description of the drawings]

[0020] [Figure 1A] Pou4f2 and / or Islet1 stimulate the regeneration of RGC-like neurons. (1A) Schematic diagram showing the transgenic constructs used to induce Ascl1 and Pou4f2 / Islet1 specifically in MG. Pou4f2 / Islet1 is surrounded by mutually exclusive floxed sites, resulting in expression of Pou4f2, Islet1, or both in the presence of active Cre. [Figure 1B] (1B) Experimental paradigm for inducing retinal regeneration in adult mice. Tamoxifen (TMX). [Figure 1C] (1C) Representative sections of retina following intravitreal NMDA injury showing transgenic expression of Pou4f2 / Brn3 and / or Islet1 in GFP+ lineage-traced MG. DAPI, 4',6-diamidino-2-phenylindole. [Figure 1D] (1D) Quantification of the percentage of transgene-expressing MG expressing Pou4f2, Islet1, or both. [Figure 1E](1E and 1F) Representative sections showing MG-derived neurons after the regeneration paradigm expressing HuC / D. [Figure 1F] (1E and 1F) Representative sections showing MG-derived neurons after the regeneration paradigm expressing HuC / D. [Figure 1G] (1G) Quantification of the percentage of GFP+ MG-derived neurons expressing either HuC / D or Otx2. [Figure 1H] (1H) Example of MG-derived neurons expressing Otx2. Significance of differences was determined using an unpaired t-test (asterisk = p < 0.0001). Dots represent individual animals. Scale bar, 50 μm. ONL, external nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer. Mouse schematic created with Biorender.com. [Figure 2A] Islet1+Pou4f2+Ascl1 (IPA)-stimulated MG-derived neurons display complex neuronal morphology. (A) Retinal whole mounts stained for GFP (MG-derived cells) and Brn3. [Figure 2B] (B–E) Examples of morphology of GFP+MG-derived cells. [Figure 2C] (B–E) Examples of morphology of GFP+MG-derived cells. [Figure 2D] (B–E) Examples of morphology of GFP+MG-derived cells. [Figure 2E] (B–E) Examples of morphology of GFP+MG-derived cells. [Figure 2F] (F) MG-derived (GFP+) cells with complex neurites co-labeled with Brn3. Scale bar, 50 μm. [Figure 3A] scRNA-seq analysis of Pou4f2 / Islet1-stimulated neurons reveals molecular characteristics of RGCs. (A) UMAP plot of FACS-sorted MG-derived cells following the IPA regeneration paradigm combined with a previous scRNA-seq dataset using Ascl1 alone. [Figure 3B](B) Feature plot highlights major clusters of MG (Rlbp1), neurogenic transition (Bhlhe22), bipolar (Cabp5), photoreceptors (Rcvrn), and RGC-like cells (Elavl4 and Sox11). [Figure 3C] (C) Distribution of cells from each treatment projected onto a partitioned UMAP plot. The donut plot represents the proportion of the data set that each cluster contains. [Figure 3D] (D) Heatmap comparing scRNA-seq datasets between Ascl1 alone and IPA treatment, with selected genes associated with RGCs illustrated. [Figure 3E] (E) Retinal section stained with Satb1 (red) for MG-derived cells (GFP), and quantification shows the percentage of GFP+ cells that are Satb1+. [Figure 3F] (F) Retinal section stained with Calretinin (red) for MG-derived cells (GFP), and quantification shows the percentage of GFP+ cells that are Calretinin+. [Figure 3G] (G) UMAP of IPA-derived neurons integrated with scRNA-seq of E14 mouse retina from Clark et al. (21) revealing that IPA neurons cluster similarly to immature RGCs. Scale bar, 50 μm. [Figure 4A] Co-induction of Islet1 and Pou4f2 stimulates MG-derived RGC-like neurons in vitro. (A) Schematic diagram of the transgenic constructs for inducing IPA in all primary MGs in vitro by doxycycline. [Figure 4B] (B) Paradigm for inducing Ascl1-mediated neurogenesis in vitro. [Figure 4C] (C-E) Representative images of EdU+ MG-derived neurons expressing neuronal markers. (C) MG-derived cells expressing EdU+ (white) and Tuj1 (red). [Figure 4D](C-E) Representative images of EdU+ MG-derived neurons expressing neuronal markers. (D) MG-derived neurons expressing EdU (white), neurofilament M (NFM; red), and the GFP transgene reporter (GFP). [Figure 4E] (C-E) Representative images of EdU+ MG-derived neurons expressing neuronal markers. (E) MG-derived neurons expressing calbindin (red) co-labeled with EdU (white), DAPI, and GFP. [Figure 4F] (F-H) UMAP plots of cultured MG reprogrammed with Ascl1 (F) and IPA (G) merged with cells from the in vivo IPA regeneration model (H) as a reference. [Figure 4G] (F-H) UMAP plots of cultured MG reprogrammed with Ascl1 (F) and IPA (G) merged with cells from the in vivo IPA regeneration model (H) as a reference. [Figure 4H] (F-H) UMAP plots of cultured MG reprogrammed with Ascl1 (F) and IPA (G) merged with cells from the in vivo IPA regeneration model (H) as a reference. [Figure 4I] (I) Stacked bar plots showing the composition of neuronal clusters in each sample. BC, bipolar cells. [Figure 4J] (J) Feature plot highlighting differentially expressed genes in neuronal clusters of either reprogramming strategy. [Figure 4K] (K) Highlighted cells of the Ascl1 and IPA datasets used for downstream DGE analysis in (L). [Figure 4L] (K) Highlighted cells of the Ascl1 and IPA datasets used for downstream DGE analysis in (L). [Figure 4M] (M) Heatmap of differentially expressed genes in either Ascl1 or IPA conditions. WT, untreated cultured MG was included for baseline values. Statistics for differential gene analysis: Wilcoxon-Mann-Whitney test for significance (P<0.05). Scale bar, 50 μm. [Figure 5A] Physiological profiling of IPA-induced neurons. (A) Overview of the electrical properties of cells in this study compared to endogenous neurons, endogenous glia, and MG-derived neurons from previous regeneration protocols (4, 5, 13). Resting potential and input resistance were estimated from current clamp recordings. [Figure 5B] (B) Example responses of three cells to current (left) and voltage (right) steps. [Figure 5C] (C) Three examples of cells responding to light stimulation. [Figure 5D] (D) Examples of cells that displayed action potentials or similar events. The two left panels are responses to hyperpolarizing and depolarizing current steps from a cell that generated an apparent Na+ spike. The two right panels are responses from a cell that generated a smaller discrete event, likely a Ca2+ spike. [Figure 6A] scATAC reveals that MG remodeled chromatin adopts an RGC-like state in response to IPA treatment. (A) Combined GFP+ sorted MG and their progeny UMAPs from an in vivo regeneration paradigm with Ascl1 alone (B) or IPA treatment (C). [Figure 6B] (A) Combination of GFP+ sorted MG and their progeny UMAPs from an in vivo regeneration paradigm with Ascl1 alone (B) or IPA treatment (C). [Figure 6C] (A) Combination of GFP+ sorted MG and their progeny UMAPs from an in vivo regeneration paradigm with Ascl1 alone (B) or IPA treatment (C). [Figure 6D] (D) Coverage plot of known marker genes used to identify clusters. [Figure 6E] (E and F) chromVAR scores of Otx2 and Pou4f2 are used to highlight differences in the accessibility of each motif. [Figure 6F] (E and F) chromVAR scores of Otx2 and Pou4f2 are used to highlight differences in the accessibility of each motif. [Figure 6G](G) Scatter plot comparing accessible motifs between E14 RGCs and IPA-induced RGCs. [Figure 6H] (H) Top “GO Biological Process” results for peaks specific to E14 RGCs compared to IPA-derived RGC-like neurons. [Figure 6I] (I) Heatmap of chromVAR activity scores of top variable motifs of TFs found in the pseudotemporal lineage of E14 progenitors to RGCs. [Figure 6J] (J) Heatmap of the same chromVAR activity scores of E14 motifs plotted over pseudotime from MG to RGC-like neurons after IPA treatment. [Figure 6K] (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. [Figure 7A] Addition of Atoh1 to the IPA paradigm promotes the transition from a precursor state to differentiated neurons. (A) Schematic of the transgenic construct for expressing IPA with Atoh1 in MG. [Figure 7B] (B) Regeneration paradigm to induce IPA:Atoh1 expression in MG of injured retina. [Figure 7C] (C) Representative immunofluorescence images of regenerated neurons from IPA:Atoh1 mice showing that MG-derived neurons (GFP+) are HuC / D+ and not Otx2+. [Figure 7D] (D) Unified UMAP of FACS-sorted MG-derived cells following regeneration paradigms with either IPA:Atoh1 or IPA alone overexpression, highlighting RGC-like cells from each dataset that were subset for further comparative analysis. [Figure 7E] (E) Scatter plot highlighting differentially expressed genes between RGC-like cells in the IPA:Atoh1 and IPA-only regeneration paradigms. [Figure 7F](F) GO analysis revealed that neurodevelopmental terms, including many retinal progenitor genes, were downregulated in the IPA:Atoh1 dataset relative to IPA alone. [Figure 7G] (G) Unified UMAP of a previously generated Ascl1:Atoh1 dataset (13) and the IPA:Atoh1 data described above. Highlighting indicates RGC-like cells from each dataset that were compared in further analysis. [Figure 7H] (H) Scatter plot highlighting genes differentially expressed between RGC-like cells in the IPA:Atoh1 and Ascl1:Atoh1 regeneration paradigms. [Figure 7I] (I) Bar plot of GO terms related to neurite outgrowth enriched in IPA:Atoh1 data. Known neuronal projection genes listed are upregulated in IPA:Atoh1 relative to Ascl1:Atoh1. [Figure 8A] A subset of IPA-derived neurons originates from the proliferative MG. (8A) Experimental paradigm for labeling dividing cells during the regeneration experiment described in FIG. [Figure 8B] (8B) Representative section showing MG-derived cells (GFP+) that had previously undergone cell division (EdU+). [Figure 8C] (8C) Representative image showing that some MG-derived neurons (GFP+ / HuC / D+, upper panel) are the result of proliferative MG (EdU+ lower left panel). Scale bar is 50 μm. Abbreviations: ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer. [Figure 9A] IPA treatment is most effective in reprogramming MGs when induced prior to injury. (9A) Experimental paradigm using transgenic mice described in Figure 1 to test whether Islet1 / Pou4f2 / Ascl1 can reprogram MGs in the absence of retinal injury. [Figure 9B] (9B) Representative images of GFP+MG showing no induction of ganglionic / amacrine marker HuC / D (upper right panel) or bipolar marker Otx2 (lower right panel). [Figure 9C] (9C) Experimental paradigm to test whether induction of IPA factors after NMDA injury and TSA application can induce MG neurogenesis. [Figure 9D] (9D) Representative image of a GFP+MG showing some co-labeling of the neuronal markers HuC / D (top right) and Otx2 (bottom right). [Figure 9E] (9E) Quantification of the percentage of MG-derived cells expressing HuC / D or Otx2 before injury, after injury, or after IPA induction without injury. Scale bar is 50 μm. [Figure 10A] scRNA-seq analysis showing that Pou4f2 biases MG production towards RGC-like neurons. (10A) UMAP of cells highlighting integrated IPAs and Ascl1-only reprogrammed MGs expressing either Isl1, Pou4f2, or a combination of both. [Figure 10B] (10B) Stacked graphs quantifying the percentage of Islet1 alone, Pou4f2 alone, or Islet1 / Pou4f2 double positive cells that terminate as MG-derived bipolar neurons or RGC-like neurons. [Figure 10C] (10C) Feature plot highlighting Ascl1 expressing cells that are downregulated as they differentiate into bipolar neurons (Otx2+) or RGC-like neurons (Elavl4+). [Figure 10D] (10D) Heatmap of the top 40 differentially expressed genes between MG and MG-derived neurons found in the GO terms “axon guidance,” “axon formation,” and “axon outgrowth.” [Figure 10E] (10E) Feature plot of predicted scores from Seurat’s labeling trans using randomly sampled cells for each major retinal neuron class and MG reference datasets subsetted from mouse retinal development scRNA-seq data23. [Figure 11A] MG-derived RGCs are a stable population over time. (11A) Total UMAP of IPA-treated MG from 3-week and 6-week endpoints. [Figure 11B](11B) Segmented UMAP showing the distribution of cells in the UMAP in (a) from each time point. [Figure 11C] (11C) Stacked graphs showing the percentage of each cluster of MG and MG-derived neurons from 3 and 6 weeks. [Figure 11D] (11D) Analysis of cell death genes found in MG-derived RGC-like neurons. These data show that cell death genes are not significantly increased in MG-derived RCS over this 3-6 week survival period. [Figure 12A] MG from IPA mice express reprogramming factors. (12A) Immunofluorescence of IPA MG treated with doxycycline for 5 days shows co-labeling of Ascl1-IRES-GFP, Brn3, and Isl1 in most cells. [Figure 12B] (12B) UMAP of untreated cultured MG labeling glial and neuronal populations. [Figure 12C] (12C) Markers of MG and various retinal neurons reveal that small populations of bipolar and amacrine cells survived the dissociation. [Figure 12D] (12D) UMAP of the combined dataset from Ascl1 overexpression in vitro and IPA overexpression in vivo and in vitro. [Figure 12E] (12E) Expression of melanopsin (Opn4) in the three datasets characterized in (12D). Scale bar is 50 μm. Abbreviations: MG, Müller glia; AC, amacrine cells; BC, bipolar cells; PR, photoreceptors; RGC, retinal ganglion cells. [Figure 13A] (13A) UMAP plot of scATAC-seq from E14 embryonic mouse retina. Clonally bar scores indicate the accessibility of motifs used to identify clusters of precursors (13B, Sox2), retinal ganglion cells (13C, Pou4f2), cones (13D, Otx2), and neurogenic precursors (13E, Ascl1). [Figure 13B](13A) UMAP plot of scATAC-seq from E14 embryonic mouse retina. Clonally bar scores indicate the accessibility of motifs used to identify clusters of precursors (13B, Sox2), retinal ganglion cells (13C, Pou4f2), cones (13D, Otx2), and neurogenic precursors (13E, Ascl1). [Figure 13C] (13A) UMAP plot of scATAC-seq from E14 embryonic mouse retina. Clonally bar scores indicate the accessibility of motifs used to identify clusters of precursors (13B, Sox2), retinal ganglion cells (13C, Pou4f2), cones (13D, Otx2), and neurogenic precursors (13E, Ascl1). [Figure 13D] (13A) UMAP plot of scATAC-seq from E14 embryonic mouse retina. Clonally bar scores indicate the accessibility of motifs used to identify clusters of precursors (13B, Sox2), retinal ganglion cells (13C, Pou4f2), cones (13D, Otx2), and neurogenic precursors (13E, Ascl1). [Figure 13E] (13A) UMAP plot of scATAC-seq from E14 embryonic mouse retina. Clonally bar scores indicate the accessibility of motifs used to identify clusters of precursors (13B, Sox2), retinal ganglion cells (13C, Pou4f2), cones (13D, Otx2), and neurogenic precursors (13E, Ascl1). [Figure 13F] (13F) Pseudotime subset of retinal progenitor cell to retinal ganglion cell transitions further analyzed in Figure 6 . [Figure 14A] Addition of Atoh1 to IPA significantly induces MG-derived RGC-like cells and does not require retinal damage. (14A) Transgenic mouse constructs used for induction of Ascl1, Atoh1, Pou4f2, and Islet1. [Figure 14B](14B) Paradigm to assay whether induction of all four transcription factors can induce MG neurogenesis in the absence of retinal injury. [Figure 14C] (14C) Representative section of retina after IPAA treatment showing that MG-derived (GFP+) cells express the neuronal marker HuC / D. [Figure 14D] (14D) Quantification of the percentage of MG-derived cells expressing the RGC-like marker HuC / D or the bipolar marker Otx2 after IPAA treatment without injury. [Figure 14E] (14E-14H) Violin plots of markers used to define the clusters and bar plots showing the cluster composition of the integrated scRNAseq data for IPA-IPA:Atoh1 (14E-14F) and Ascl1:Atoh1-IPA:Atoh1 (14G-14H). [Figure 14F] (14E-14H) Violin plots of markers used to define the clusters and bar plots showing the cluster composition of the integrated scRNAseq data for IPA-IPA:Atoh1 (14E-14F) and Ascl1:Atoh1-IPA:Atoh1 (14G-14H). [Figure 14G] (14E-14H) Violin plots of markers used to define the clusters and bar plots showing the cluster composition of the integrated scRNAseq data for IPA-IPA:Atoh1 (14E-14F) and Ascl1:Atoh1-IPA:Atoh1 (14G-14H). [Figure 14H] (14E-14H) Violin plots of markers used to define the clusters and bar plots showing the cluster composition of the integrated scRNAseq data for IPA-IPA:Atoh1 (14E-14F) and Ascl1:Atoh1-IPA:Atoh1 (14G-14H). [Figure 15]Promoting RGC production using transcription factors specific for developing retinal cells. Lentivirus was used to induce the expression of several transcription factors 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. Of all factors tested, Onecut1 (arrow) was able to induce Müller glia to generate new cells with gene expression characteristic of RGCs. [Figure 16] Promoting RGC production using transcription factors specific for developing retinal cells. Expression of Onecut1 (in FIG. 15) induced Müller glia to generate new cells with gene expression characteristic of RGCs, including Nefm, Nefl, Ebf1, and Gap43. [Figure 17A] Reprogramming transcription factors delivered by AAV can induce neurogenesis. HuC / D+ neuronal lineage was traced from MG by tdTomato. (17A) AAV design using Atoh1. [Figure 17B] (17B) Protocol for lineage tracing. [Figure 17C] (17C) Immunofluorescence demonstration of successful delivery of reprogramming transcription factors resulting in reprogrammed HuC / D+ neurons from MG cells. [Figure 18] Reprogramming efficiency in lineage traced MG. Bar graph showing that both Atoh1 and Atoh7 delivered by AAV can induce HuC / D+ neurons. Atoh1 and Atoh7 have similar efficiency. AAV delivery of reprogramming factors under these conditions was much less efficient than transgenic expression. [Figure 19] Irx2 and Neurod2 promote axonal growth in IPA-reprogrammed MG. [Figure 20A]Human Müller glia generated in vitro from fetal retina or pluripotent stem cells using retinal spheres and retinal organoids. (20A) Schematic of the embryonic stem cell (ESC) differentiation protocol to generate retinal organoids (RO). [Figure 20B] (20B) Images of MG development over time in retinal organoids labeled with RLBP1 and SOX2. [Figure 20C] (20C) The top panel shows a schematic protocol for the generation of retinospheres (RS), and the bottom panel shows images of retinospheres generated from multiple fetal retinas and cultured for various times as labeled. [Figure 20D] (20D) Characterization of MG in RS using RLBP1, VSX2, SOX9, SOX2, and GFAP. [Figure 21A] A protocol for generating dissociated human Müller glia cultures in vitro. [Figure 21B] A protocol for generating dissociated human Müller glia cultures in vitro. [Figure 22A] Ascl1 promotes neurogenesis in human Müller glia. (22A) Protocol for delivery of GFP into dissociated cells. [Figure 22B] (22B-22E) These neurons express several pan-neuronal markers, such as DCX and TUJ1, but are not mature. [Figure 22C] (22B-22E) These neurons express several pan-neuronal markers, such as DCX and TUJ1, but are not mature. [Figure 22D] (22B-22E) These neurons express several pan-neuronal markers, such as DCX and TUJ1, but are not mature. [Figure 22E] (22B-22E) These neurons express several pan-neuronal markers, such as DCX and TUJ1, but are not mature. [Figure 23] Characterization of ShH10 capsid and RLBP promoters in NHP dissociated Müller glia cultures. [Figure 24] HES1 promoter as an alternative to the RLBP1 promoter. Schematic diagram of the lentiviral construct containing the HES1 promoter driving the expression of EGFP. [Figure 25A] Schematic diagram of the construction of lentiviruses containing the HES1 promoter driving expression of EGFP (control) or the proneural factor ASCL1 and EGFP in Müller glia (25A). [Figure 25B] (25B-25D) Testing the HES1 promoter for specificity of MG expression in human retinal spherules (as of infection day D132+77=D209). [Figure 25C] (25B-25D) Testing the HES1 promoter for specificity of MG expression in human retinal spherules (as of infection day D132+77=D209). [Figure 25D] (25B-25D) Testing the HES1 promoter for specificity of MG expression in human retinacules (at infection day D132+77=D209). GFP and HES1 are found in the same cells. [Figure 26] The HES1 promoter also directs expression in MG in adult NHP dissociated cultures. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0021] The molecules, compositions, and methods described herein are based on the surprising discovery that retinal ganglion-like cells can be regenerated in damaged adult retinas in vivo by targeted overexpression of developmental retinal ganglion cell transcription factors. As demonstrated herein, AAV vectors can deliver reprogramming transcription factors to Müller glia in vivo. Although the rate of neurogenesis from AAV-reprogrammed Müller glia is much lower than that observed with transgenic reprogramming, other RGC transcription factors can increase specific RGC genes and, in some instances, improve RGC axon growth. Furthermore, in the studies described herein, human Müller glia can be reprogrammed to generate neurons in dissociated cultures, and these human Müller glia can be derived from either retinal organoids or fetal human retina. This ability to reprogram Müller glia to specific types is particularly important for endogenous regeneration strategies, since most blinding diseases are the result of defects in specific neuronal subtypes. For example, glaucoma is primarily caused by the death of RGCs.

[0022] definition All scientific and technical terms used in this application have the meanings commonly used in the art unless otherwise specified. As used in this application, the following words or phrases have the meanings specified:

[0023] As used herein, the term "comprising" is intended to mean that the compositions and methods include the recited elements but do not exclude other elements. As used herein, the transitional phrase "consisting essentially of" (and grammatical equivalents) shall be construed to include the recited materials or steps and materials or steps that do not materially affect the basic and novel feature(s) of the recited embodiment. Thus, as used herein, the term "consisting essentially of" should not be construed as equivalent to "comprising." "Consisting of" shall mean excluding more than trace amounts of other component elements and other substantial method steps for administering the compositions disclosed herein. The aspects defined by each of these transitional phrases are within the scope of this disclosure.

[0024] As used herein, "retinal neurons" refer 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.

[0025] As used herein, the term "nucleic acid sequence" or "polynucleotide" refers to nucleotides of any length that are deoxynucleotides (i.e., DNA) or derivatives thereof; ribonucleotides (i.e., RNA) or derivatives thereof; or peptide nucleic acid (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 naturally occurring, synthetic, modified, mutated, or non-DNA or RNA naturally occurring forms.

[0026] MicroRNAs, or "miRNAs," or "miRs," are short non-coding RNAs that regulate gene expression through post-transcriptional regulation of target genes.

[0027] "Short hairpin RNA" or "shRNA" is a synthetic or non-naturally occurring RNA molecule. shRNA refers to an RNA with a tight hairpin turn that is used to silence the expression of a target gene in a cell (via RNA interference or RNAi). shRNAs are typically delivered via an expression vector, such as a DNA plasmid, or a viral vector.

[0028] The term "vector" refers to, but is not limited to, a recombinant genetic construct or plasmid, or an expression construct or expression vector, that retains the ability to infect and transduce non-dividing and / or slowly dividing cells and integrate into the genome of a target cell. A vector can be derived from or based on a wild-type virus. Aspects of the present disclosure relate to adeno-associated virus vectors, adenoviral vectors, and lentiviral vectors.

[0029] The term "expression control element" as used herein refers to any sequence that controls the expression of a coding sequence such as a gene. Exemplary expression control elements include, but are not limited to, promoters, enhancers, microRNAs, post-transcriptional regulators, polyadenylation signal sequences, and introns. Expression control elements can be, but are not limited to, constitutive, inducible, repressible, or tissue-specific. A "promoter" is a control sequence that is a region of a polynucleotide sequence where the initiation and rate of transcription is controlled. It can include genetic elements to which regulatory proteins and molecules can bind, such as RNA polymerase and other transcription factors. In some embodiments, expression control by a promoter is tissue-specific. An "enhancer" is a region of DNA that can be bound by an activating protein to increase the likelihood or frequency of transcription. Non-limiting exemplary enhancers and post-transcriptional regulators include the CMV enhancer and the WPRE.

[0030] The term "multicistronic" or "polycistronic" or "bicistronic" or "tricistronic" refers to an mRNA that has multiple, i.e., duplicated or tripled, coding regions or exons, and thus has the ability to obtain two or more, or three or more, or four or more, etc., proteins from an mRNA from a single construct. A multicistronic vector simultaneously expresses two or more separate proteins from the same mRNA. The two most widely used strategies to construct a multicistronic construct are by using 1) an IRES, or 2) a 2A self-cleaving site. "IRES" refers to an internal ribosome entry site or a portion thereof of viral, prokaryotic, or eukaryotic origin that is used within a polycistronic vector construct. In some embodiments, an IRES is an RNA element that allows translation initiation 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 linking sequence used within a vector construct to incorporate a site that facilitates ribosomal skipping, and thus generates 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.

[0031] The term "substantially complementary", when used to define either an amino acid sequence or a nucleic acid sequence, means that a particular sequence, e.g., an oligonucleotide sequence, is substantially complementary to the sequence referred to. Thus, typically, a sequence is highly complementary to a "target" sequence, with no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 base mismatches throughout the sequence. In many cases, it may be desirable for a sequence to be perfectly matched, i.e., completely complementary to the sequence to which the nucleic acid specifically binds, and thus have zero mismatches along the complementary stretch. Thus, highly complementary sequences usually bind very specifically to the target sequence region, and are therefore very efficient in reducing and / or inhibiting the biological activity of the target sequence.

[0032] A substantially complementary nucleic acid sequence is about 80 percent complementary (or "perfect match") to the corresponding target sequence to which the nucleic acid specifically binds, and more preferably, is greater than about 85 percent 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 invention, in which case the nucleic acid sequence is greater than about 90 percent complementary to the corresponding target sequence to which the nucleic acid specifically binds, and in certain embodiments may be greater than about 95 percent complementary to the corresponding target sequence to which the nucleic acid specifically binds, up to 96%, 97%, 98%, 99%, or even 100% perfectly complementary to the corresponding target sequence to which the nucleic acid specifically binds.

[0033] "Homology" or "identity" or "similarity" refers to sequence similarity between two peptides or two nucleic acid molecules. Homology can be determined by comparing positions in each sequence that can be aligned for comparison purposes. If a position in the compared sequence is occupied by the same base or amino acid, the molecules are homologous at that position. The degree of homology between sequences is a function of the number of matching or homologous positions shared by the sequences. An "unrelated" or "non-homologous" sequence shares less than 40% identity, or alternatively less than 25% identity, with one of the sequences disclosed herein.

[0034] Percent similarity or percent complementarity of any of the sequences of the present disclosure can be determined by comparing sequence information using, for example, the GAP computer program, version 6.0, available from the University of Wisconsin Genetics Computer Group (UWGCG). The GAP program utilizes 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. Preferred default parameters of the GAP program include: (1) a one-way comparison matrix of nucleotides (containing values ​​of 1 for identity and 0 for non-identity) and a weighted comparison matrix of Gribskov and Burgess (1986); (2) a penalty of 3.0 for each gap and an additional penalty of 0.10 for each symbol in each gap; and (3) no penalty for end gaps.

[0035] "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, to provide a sequence that can specifically bind to a target molecule and act as an antisense construct to alter, reduce, or inhibit the biological activity of the target.

[0036] As used herein, in the context of antisense oligonucleotides, "directed against" means that the antisense oligonucleotide binds to a target miRNA and blocks or inhibits the activity of the target.

[0037] As used herein, the terms "protein," "peptide," and "polypeptide" refer to amino acid subunits, amino acid analogs, or peptidomimetics. The subunits may be linked by peptide bonds. In alternative embodiments, the subunits may be linked by other bonds, e.g., esters, ethers, etc. As used herein, the term "amino acid" refers to either natural and / or unnatural or synthetic amino acids.

[0038] As used herein, the term "recombinant expression system" or "recombinant expression vector" refers to a genetic construct for expressing certain recombinantly formed genetic material.

[0039] The term "effective amount" or "therapeutically effective amount" or "prophylactically effective amount" refers to an amount of an active agent described herein effective to produce 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 produce regeneration of retinal neurons and / or to improve or ameliorate the symptoms of a retinal degenerative disease and / or to treat a retinal degenerative disease.

[0040] When the disclosure of this specification relates to a small molecule, a polypeptide, a protein, a polynucleotide, a nucleic acid, an oligonucleotide, an antisense, or an miRNA, the equivalent or biological equivalent of such is intended to be within the scope of this disclosure. As used herein, the term "biological equivalent thereof" is intended to be synonymous with "equivalent thereof" when referring to a reference small molecule, a polypeptide, a protein, a polynucleotide, a nucleic acid, an oligonucleotide, an antisense, or an miRNA, even if the reference molecule has minimal homology while maintaining a desired structure or function. Unless specifically recited herein, any nucleic acid, polynucleotide, oligonucleotide, antisense, miRNA, polypeptide, or protein referred to herein is also intended to include their equivalent. For example, an equivalent is at least about 70% homology or identity, or at least 80% homology or identity, or at least about 85%, or alternatively at least about 90%, or alternatively at least about 95%, or alternatively 98% percent homology or identity, and exhibits substantially the same biological activity as the reference protein, polypeptide, or nucleic acid. Alternatively, when referring to a polynucleotide, the equivalent is a polynucleotide that hybridizes under stringent conditions to the reference polynucleotide or its complement.

[0041] In some embodiments disclosed herein, polypeptide and / or polynucleotide sequences are provided herein for use in gene and protein introduction and expression techniques described below. Such sequences provided herein can be used to provide expression products, as well as substantially identical sequences that generate proteins with the same biological properties. These "biologically equivalent" or "biologically active" or "equivalent" polypeptides are encoded by equivalent polynucleotides described herein. They have a primary amino acid sequence that is at least 60%, or alternatively at least 65%, or alternatively at least 70%, or alternatively at least 75%, or alternatively at least 80%, or alternatively at least 85%, or alternatively at least 90%, or alternatively at least 95%, or alternatively at least 98% identical to a reference polypeptide when compared using sequence identity methods performed under default conditions. Specific polynucleotide or polypeptide sequences are provided as examples of specific embodiments. Modifications may be made to the amino acid sequence by using another amino acid with a similar charge. Furthermore, an equivalent polynucleotide is one that hybridizes under stringent conditions to a reference polynucleotide or its complementary strand, or, in the case of a polypeptide, a polypeptide encoded by a polynucleotide that hybridizes under stringent conditions to a reference coding polynucleotide or its complementary strand. Alternatively, an equivalent polypeptide or protein is one that is expressed from an equivalent polynucleotide.

[0042] "Hybridization" refers to a reaction in which one or more polynucleotides react to form a complex stabilized through hydrogen bonds between the bases of nucleotide residues. The hydrogen bonds may occur by Watson-Crick base pairing, Hoogsteen binding, or any other sequence-specific method. The complex may include two strands forming a duplex structure, three or more strands forming a multistranded complex, a self-hybridizing single strand, or any combination of these. A hybridization reaction may constitute a step in a larger process, such as the initiation of a PC reaction, or the enzymatic cleavage of a polynucleotide by a ribozyme.

[0043] 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 wash 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 wash 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 wash solution of about 1xSSC, 0.1xSSC, or deionized water. Generally, hybridization incubation times are between 5 minutes and 24 hours, with one, two, or more washing steps, with wash incubation times of about 1, 2, or 15 minutes. SSC is a 0.15M NaCl and 15 mM citrate buffer. It will be understood that equivalents of SSC using other buffer systems may be used.

[0044] As used herein, "treating" a subject with a retinal degenerative disease or "treatment" of a retinal degenerative disease refers to: 1) preventing the onset of a disease or disorder in a subject who is susceptible to the disease or who has not yet exhibited symptoms of the disease; 2) inhibiting or arresting the development of the disease; or 3) alleviating or causing regression of the disease or symptoms of the disease.As is well understood in the art, "treatment" is an approach to obtain beneficial or desired results, including clinical results. For purposes of the compositions, combination therapies, nucleic acid molecules, and methods disclosed herein for inducing neurogenesis from MG and / or generating functional neurons from MG, beneficial or desired results may include, but are not limited to, one or more of the following: alleviation or amelioration of one or more symptoms of retinal degeneration, whether detectable or undetectable; reduction in the extent of a retinal degenerative pathology (including a retinal degenerative disease); stable state (i.e., not worsening) of a retinal degenerative pathology (including a disease); delayed or slowed retinal degenerative pathology (including a disease); progression, amelioration, or alleviation of a retinal degenerative pathology (including a disease); and status and remission (partial or total) of retinal degeneration, whether detectable or undetectable.

[0045] As used herein, the term "isolated" means that a naturally occurring DNA fragment, DNA molecule, coding sequence, or oligonucleotide is removed from its natural environment or is a synthetic molecule or cloning product. Preferably, the DNA fragment, DNA molecule, coding sequence, or oligonucleotide is purified, i.e., essentially free from any other DNA fragments, DNA molecules, coding sequences, or oligonucleotides and associated cellular products or other impurities.

[0046] The term "cell" as used herein refers to either a prokaryotic or 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 a human, non-human animal, mammal, or non-human mammal, including, but not limited to, cells of a 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 glia (MG), and / or retinal neural cells, such as retinal neurons, bipolar neurons, amacrine cells, horizontal cells, ganglion cells, and / or glia. The term "Müller glia" cells, or "Müller glia" or "MG" refers to cells found in the vertebrate retina and are support cells for neurons. MGs are the most common type of glial cell in the retina. MG cell bodies are located in the inner nuclear layer of the retina, but MGs span the entire retina.

[0047] As used herein, the term "subject" includes any human or non-human animal. The term "non-human animal" includes all vertebrates, e.g., mammals and non-mammals, such as non-human primates, horses, sheep, dogs, cows, pigs, chickens, and other veterinary subjects.

[0048] As used herein, "a" or "an" means at least one, unless clearly indicated otherwise.

[0049] As used herein, "preventing" or "protecting against" a condition or disease means to hinder, reduce, or delay the onset or progression of a condition or disease.

[0050] The term "encode" as applied to a nucleic acid sequence refers to a polynucleotide that, in its natural state or when engineered by methods well known to those of skill in the art, is said to "encode" a polypeptide, mRNA, or effector RNA, and is capable of transcribing and / or translating the effector RNA, mRNA, or mRNA which can lead to a polypeptide and / or fragments thereof. The antisense strand is the complementary strand of such a nucleic acid, from which a coding sequence can be deduced.

[0051] As used herein, the term "expression" or "gene expression" refers to the process by which a polynucleotide is transcribed into mRNA and / or the transcribed mRNA is subsequently translated into a peptide, polypeptide, or protein. If the polynucleotide is derived from genomic DNA, expression may include splicing of the mRNA in eukaryotic cells. The expression level of a gene may be determined by measuring the amount of mRNA or protein in a cell or tissue sample; furthermore, the expression levels of multiple genes may be determined to establish an expression profile of a particular sample.

[0052] As used herein, the term "functional" may be used to modify any molecular, biological, or cellular material with the intent of achieving a particular, specified effect.

[0053] As used herein, the term "combination therapy" refers to two or more compositions and / or nucleic acid molecules that are delivered in combination, for example, but not limited to, sequentially, concurrently, simultaneously, and / or stepwise, to achieve a therapeutic effect.

[0054] The term "about" as used herein when referring to a measurable value such as an amount, level, or concentration is meant to encompass, for example, but not limited to, a 20%, 10%, 5%, 1%, 0.5%, or even 0.1% variation of a stated amount, or a quantifiable fold difference in level of comparison to a standard or control or reference material, including, for example, 1-fold, 2-fold, 3-fold, 4-fold... 10-fold, 100-fold, etc. of a particular level of comparison.

[0055] In some embodiments, enhancing the expression levels of two or more endogenous and / or exogenous proneural bHLH transcription factors refers to increased expression of endogenous and / or exogenous Ascl1, and / or Atoh1, and / or Atoh7, including but not limited to, as compared to levels in untreated or Ascl1-expressing control samples or explants alone. In some embodiments, increased neurogenesis and / or increased production of functional neurons as compared to controls. In some embodiments, expression levels and / or functional neurons are increased by about 1.1 fold, about 1.2 fold, about 1.3 fold, about 1.4 fold, about 1.5 fold, about 1.6 fold, about 1.7 fold, about 1.8 fold, about 1.9 fold, about 2 fold, about 2.5 fold, about 3 fold, about 4 fold, about 5 fold, about 6 fold, about 7 fold, about 8 fold, about 9 fold, about 10 fold, about 20 fold, about 50 fold, about 100 fold, about 1000 fold, or about 10,000 fold as compared to a control.

[0056] In some embodiments, the terms "reprogramming potentiator" or "reprogramming potentiating agent", as used interchangeably herein, refer to a small molecule, polypeptide, protein, polynucleotide, nucleic acid, oligonucleotide, antisense, miRNA, or equivalent or biological equivalent thereof, which provides a composition that supports the process of stimulating and / or enhancing neurogenesis from MGs in such a way that functional neurons from MGs are produced. In one embodiment, the one or more reprogramming enhancing agents stimulate neurogenesis by inhibiting the HDAC pathway, supporting the process of producing functional neurons from MGs. In another embodiment, the one or more reprogramming enhancing agents stimulate neurogenesis by inhibiting the Jak / STAT pathway, supporting the process of producing functional neurons from MGs. In another embodiment, the one or more reprogramming enhancing agents stimulate neurogenesis by inhibiting the HDAC+Jak / STAT pathway, supporting the process of producing functional neurons from MGs. In another embodiment, the one or more reprogramming enhancers stimulate neurogenesis by enhancing and / or increasing endogenous and / or exogenous Ascl1 expression levels, supporting the process of producing functional neurons from MG. See also our prior work in WO2019 / 210320 and WO2020 / 223308, each of which is incorporated herein by reference in its entirety.

[0057] The terms "acceptable," "effective," or "sufficient," when used to describe the selection of any components, ranges, dosage forms, etc. disclosed herein, are intended to mean that said components, ranges, dosage forms, etc. are suitable for the purpose disclosed.

[0058] The term "adeno-associated virus" or "AAV" as used herein refers to a member of a class of viruses related to this name and belonging to the genus dependoparvovirus of the family Parvoviridae. Multiple serotypes of this virus are known to be suitable for gene delivery, and all known serotypes are capable of infecting cells of various tissue types. At least 11 or 12, numbered consecutively, have been disclosed in the prior art. Non-limiting exemplary serotypes useful in the methods disclosed herein include any of the 11 or 12 serotypes, e.g., AAV2, AAV5, and AAV8, or engineered serotypes, e.g., AAV-SHH10 and AAV-7m8. AAV structural particles are composed of 60 protein molecules composed of VP1, VP2, and VP3. Each particle contains approximately 5 VP1 proteins, 5 VP2 proteins, and 50 VP3 proteins ordered into an icosahedral structure.

[0059] Nucleic Acid Molecules and Compositions Compositions and / or nucleic acid molecules are provided for regenerating the retina, enhancing retinal regeneration, stimulating the regeneration of retinal ganglion cells, restoring vision, and treating retinal degenerative disease, damage, or injury.

[0060] 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 retroviral and lentiviral delivery. An example of a non-viral delivery method is cell penetrating peptides (CPPs). Polynucleotide constructs may also be modified, such as by chemical modification, to improve their stability and / or suitability for delivery. In some embodiments, oligonucleotides are modified by locked nucleic acid and / or phosphorothioate linkages. In some embodiments, delivery systems are selected for improved bioavailability, such as, for example, PEGylated liposomes, lipidoids, or biodegradable polymers.

[0061] In some embodiments, the composition further comprises one or more additional enhancing or therapeutic agents, including, for example, a reprogramming enhancing agent. In some embodiments, the composition does not comprise a reprogramming enhancing agent. Optionally, a composition comprising one or more small molecule reprogramming enhancing agents can be administered sequentially or simultaneously with the nucleic acid molecules disclosed herein. In another embodiment, one or more protein / peptide or miR-based reprogramming enhancing agents can be incorporated into the nucleic acid molecules disclosed herein. Such one or more reprogramming enhancing agents are selected from HDACi, STATi, Jak / STATi, and RNAi-based Ascl1 activators. See also our prior work in WO2019 / 210320 and WO2020 / 223308, each of which is incorporated herein by reference in its entirety.

[0062] 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), (Pro) / Istodax™ also known as Romidepsin, (Pro) / Beleodaq™ also known as Belinostat, (Pro) / Farydak™ also known as Panobinostat, and (Pro) / Zolinza™ also known as Vorinostat, Quisinostat, Abexinostat, Givinostat, Resminostat, Phenylbutyrate, ValproicAcid, Depsipeptide, Entinostat, Mocetinostat, and Tubastatin A. [7] Exemplary HDACi peptides are, but are not limited to, 16cyc-HxA, 16lin-HxA, and 16KA.

[0063] In some embodiments, the inhibitor, mimetic, activator, or antagomir is an oligonucleotide or nucleotide sequence.Thus, the invention provides nucleotide constructs for use in the compositions or combination therapies or nucleic acid molecules and methods described herein.

[0064] In some embodiments, the reprogramming enhancer is selected from one or more STAT signaling pathway inhibitors, 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.

[0065] Optionally, the composition is provided, comprising any one or more of RNAi-based Ascl1 activator and / or combination therapy with HDACi+STATi, and / or nucleic acid sequence encoding developmental RGC transcription factor, or vector comprising nucleic acid sequence provided herein, and carrier.In some embodiments, carrier is a pharmaceutically acceptable carrier.

[0066] An exemplary nucleic acid sequence encoding human Onecut1 can be found in NCBI Reference SEQ ID NO: NC_000015.10 (SEQ ID NO: 1). An exemplary nucleic acid sequence encoding human Pou4f2 can be found in NCBI Reference SEQ ID NO: NC_000004.12 (SEQ ID NO: 3). An exemplary nucleic acid sequence encoding human Islet1 can be found in NCBI Reference SEQ ID NO: NC_000005.10 (SEQ ID NO: 5). An exemplary nucleic acid sequence encoding human Irx2 can be found in NCBI Reference SEQ ID 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 the amino acid sequence of SEQ ID NO: 65). An exemplary nucleic acid sequence encoding human Irx5 can be found in NCBI Reference SEQ ID 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 the amino acid sequence of SEQ ID NO: 67). An exemplary nucleic acid sequence encoding Neurod2 can be found in NCBI Reference Sequence: NC_000017.11 (SEQ ID NO: 17). An exemplary nucleic acid sequence encoding human Ebf1 can be found in NCBI Reference Sequence: 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 the amino acid sequence of SEQ ID NO: 69). An exemplary nucleic acid sequence encoding human Tcf3 can be found in NCBI Reference Sequence: 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 the amino acid sequence of SEQ ID NO: 71). This mouse sequence can be used to guide the selection of the corresponding human or humanized Tcf3.

[0067] An exemplary nucleic acid sequence encoding human Ascl1 can be found in NCBI reference sequence 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. Included herein are Ascl1 homologs, including, but not limited to, those from species such as mouse, dog, horse, etc.

[0068] The protein Atonal homolog 1 (Atoh1) is a proneural member of the bHLH transcription factor family. This protein activates an E-box distinct from the Ascl1 gene. An exemplary nucleic acid sequence encoding human Atoh1 can be found in NCBI reference sequence 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 Atoh1 homologs, orthologs, and / or paralogs from species such as, but not limited to, mouse, dog, horse, etc.

[0069] The Atoh7 family bHLH transcription factor 7 (Atoh7) gene encodes a proneural member of the basic helix-loop-helix (BHLH) family of transcription factors. An exemplary nucleic acid sequence encoding human Atoh7 can be found in NCBI Reference SEQ ID NO: NM_008553.4 (SEQ ID NO: 54). In another embodiment, disclosed herein is a nucleic acid sequence encoding a human Atoh7 amino acid sequence of NP_660161.1 (SEQ ID NO: 55), or a portion thereof. Included herein are Atoh7 homologs, orthologs, and / or paralogs from species such as, but not limited to, mouse, dog, horse, etc. An exemplary nucleic acid sequence encoding Neurogenin-2 (also known as NEUROG2 and NGN-2) can be found in NCBI Reference SEQ ID NO: NM_024019. An exemplary nucleic acid sequence encoding Neurod1 can be found in NCBI Reference SEQ ID NO: KR709666.

[0070] Exemplary nucleic acid sequences of Ascl1, Atoh1 and / or Atoh7 or other proneural bHLH transcription factors for use herein include, but are not limited to, portions of the corresponding Ascl1, Atoh1 and / or Atoh7 sequences for purposes of multicistronic, bicistronic and / or tricistronic constructs, plasmids and / or expression vectors, It is understood that portions of the sequences mentioned herein can be selected for use, and that the selected portions are sufficient to encode the listed transcription factor(s) and / or other factors.

[0071] Exemplary 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 NM_004172). Viral Vectors

[0072] In some embodiments, the vector is a viral vector. In some embodiments, the vector is an adenoviral vector, an adeno-associated virus (AAV), or a lentiviral vector. In some embodiments, the vector is a retroviral vector, an adenoviral / retroviral chimeric vector, a herpes simplex virus I or II vector, a parvoviral vector, a reticuloendotheliosis virus vector, a poliovirus vector, a papillomavirus vector, a vaccinia virus vector, or any hybrid or chimeric vector incorporating preferred aspects of two or more viral vectors. In some embodiments, the vector further comprises one or more expression control elements operably linked to the polynucleotide. In some embodiments, the vector further comprises one or more selection markers.

[0073] In some embodiments, the vector disclosed herein is an AAV vector with low toxicity.In some embodiments, the AAV vector does not integrate into the host genome, and therefore has a low probability of causing insertion mutation.In some embodiments, the AAV vector can encode a total polynucleotide range of 4.5kb to 4.75kb. In some embodiments, exemplary AAV vectors that may be used in any of the compositions, systems, methods, and kits described herein include an AAV1 vector, a modified AAV1 vector, an AAV2 vector, a modified AAV2 vector, an AAV3 vector, a modified AAV3 vector, an AAV4 vector, a modified AAV4 vector, an AAV5 vector, a modified AAV5 vector, an AAV6 vector, a modified AAV6 vector, an AAV7 vector, a modified AAV7 vector, an AAV8 vector, an AAV9 vector, an AAV.rh10 vector, a modified AAV.rh10 vector, an AAV.rh32 / 33 vector, a modified AAV.rh32 / 33 vector, an AAV.rh43 vector, a modified AAV.rh43 vector, an AAV.rh64R1 vector, and a modified AAV.rh64R1 vector, and any combination or equivalent thereof.

[0074] 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, lentiviral vector may refer to transgene plasmid vectors and transgene plasmid vectors, along with associated plasmids (e.g., packaging plasmids, rev expression plasmids, envelope plasmids), as well as lentivirus-based particles that can introduce exogenous nucleic acid into cells via a viral or viral-like entry mechanism. Lentiviral vectors are well known in the art. In some embodiments, exemplary lentiviral vectors that may be used in connection with any of the compositions, nucleic acid molecules, and / or methods described herein 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, soot-borne immunodeficiency virus (SIV) vectors, and the like. SM ) vector, modified mangabenician immunodeficiency virus (SIV) SM ) vector, African green monkey immunodeficiency virus (SIV) AGM ) vector, modified African green monkey immunodeficiency virus (SIV AGM ) vector, an Equine Infectious Anemia Virus (EIAV) vector, a modified Equine Infectious Anemia Virus (EIAV) vector, a Feline Immunodeficiency Virus (FIV) vector, a modified Feline Immunodeficiency Virus (FIV) vector, a Visna / Mezivirus (VNV / VMV) vector, a modified Visna / Mezivirus (VNV / VMV) vector, a Caprine Arthritis Encephalopathy Virus (CAEV) vector, a modified Caprine Arthritis Encephalitis Virus (CAEV) vector, a Bovine Immunodeficiency Virus (BIV), or a modified Bovine Immunodeficiency Virus (BIV).

[0075] 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 isolated or derived from a retrovirus. In some embodiments, the viral vector comprises a sequence isolated or derived from a lentivirus. In some embodiments, the viral vector comprises a sequence isolated or derived from an adenovirus. In some embodiments, the viral vector comprises a sequence isolated or derived from an adeno-associated virus (AAV). In some embodiments, the viral vector is replication-incompetent. In some embodiments, the viral vector is isolated or recombinant. In some embodiments, the vector is self-complementary.

[0076] In some embodiments of the compositions and / or nucleic acid molecules and / or methods of the present disclosure, the viral vector comprises sequences isolated or derived from adeno-associated virus (AAV). In some embodiments, the viral vector comprises inverted terminal repeat or capsid sequences isolated or derived from serotypes AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, or AAV12, or the vector and / or components are derived from synthetic AAV serotypes, such as, but not limited to, Anc80 AAV (the ancestor of AAV1, 2, 6, 8, and 9). In some embodiments, the viral vector is replication-incompetent. In some embodiments, the viral vector is isolated or recombinant (rAAV). In some embodiments, the vector is self-complementary (scAAV).

[0077] In some embodiments of the compositions and methods of the present disclosure, the vector of the present disclosure is a non-viral vector, hi some embodiments, the vector comprises or consists of a nanoparticle, a micelle, a liposome or lipoplex, a polymersome, a polyplex, or a dendrimer.

[0078] In some embodiments, the expression vector or viral vector disclosed herein is used to transfect, transform, or contact a cell that is a eukaryotic cell. In certain embodiments, the cell is an animal cell. In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is a bovine, murine, feline, equine, porcine, canine, monkey, or human cell. In certain embodiments, the cell is an animal or mammalian retinal neuron or MG.

[0079] In some embodiments, the cell is a packaging cell or a producer cell for the production of viral particles.

[0080] In some embodiments, provided herein is a viral particle 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.

[0081] In general, methods for packaging genetic material, such as RNA or DNA, into one or more vectors are well known in the art. For example, the genetic material can be packaged using packaging vectors and cell lines and introduced via conventional recombinant methods.

[0082] In some embodiments, the packaging vector may include, but is not limited to, retroviral vectors, lentiviral vectors, adenoviral vectors, and adeno-associated viral vectors. The packaging vector includes factors and sequences that facilitate the delivery of genetic material into cells. For example, the retroviral construct is a packaging plasmid that includes at least one retroviral helper DNA sequence derived from a replication-incompetent retroviral genome that encodes in trans all virion proteins required for packaging a replication-incompetent retroviral vector, for producing virion proteins capable of packaging high titers of replication-incompetent retroviral vectors without the production of replication-competent helper virus. The retroviral DNA sequence lacks the region that encodes the natural enhancer and / or promoter of the viral 5'LTR of the virus, lacks both the psi functional sequence and the 3'LTR involved in packaging the helper genome, but encodes an exogenous polyadenylation site, for example, the SV40 polyadenylation site, and an exogenous enhancer and / or promoter that directs efficient transcription in the cell type in which virus production is desired. 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 Moloney Murine Sarcoma Virus (MMSV) enhancer and promoter (U3 region), the Rous Sarcoma Virus (RSV) U3 region, the Spleen Focus Forming Virus (SFFV) U3 region, or the HCMV IE enhancer linked to the native Moloney Murine Leukemia Virus (MMLV) promoter.

[0083] A retroviral packaging plasmid may consist of two retroviral helper DNA sequences encoded by a plasmid-based expression vector, for example, the first helper sequence contains cDNA encoding the gag and pol proteins of ecotropic MMLV or GALV, and the second helper sequence contains cDNA encoding the env protein. The Env gene that determines the host range may be derived from xenotropic, amphipathic, ecotropic, polytropic (mink focus forming) or 10A1 murine leukemia virus env protein, or from 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 domains of the above env gene products, as well as genes encoding monoclonal antibodies against specific surface molecules on the desired target cells. Similar vector-based systems may use other vectors such as the Sleeping Beauty vector or transposon-based elements.

[0084] The resulting packaged expression system can then be introduced via a suitable route of administration, as discussed in detail with respect to the method aspects disclosed herein.

[0085] Pharmaceutical Compositions The pharmaceutical compositions disclosed herein may include one or more pharma- ceutical and 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, amino acids such as polypeptides or glycine, antioxidants, chelating agents such as EDTA or glutathione, adjuvants (e.g., aluminum hydroxide), and preservatives. The compositions of the present disclosure may be formulated for intraocular administration.

[0086] cell In some embodiments of the compositions and / or nucleic acid molecules and / or methods of the present disclosure, the cell of the present disclosure is a Muller glia (MG) cell, or a retinal cell, such as a rod photoreceptor cell or a cone photoreceptor cell. In some embodiments, the cell is a neuronal cell. In some embodiments, the neuronal cell of the present disclosure is a retinal neuron. In some embodiments, the neuronal cell of the present disclosure is an optic nerve neuron. In some embodiments, the neuronal cell of the present disclosure is a neuroglial or glial cell. In some embodiments, the cell is a bipolar neuron, a horizontal cell, a ganglion cell, or an amast cell. In some embodiments, the cell of the present disclosure is an astrocyte. In some embodiments, the cell of the present disclosure is a macroglia or a microglia or a glia.

[0087] In some embodiments of the compositions and methods of the present disclosure, the cells of the present disclosure are cultured cells.

[0088] 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 to the retina of a subject in need thereof.

[0089] In some embodiments, the cells of the present disclosure are autologous or allogeneic and are used for transplantation.

[0090] In some embodiments, the cell of the present disclosure is stem cell-derived or embryonic stem cell-derived retinal cell.In some embodiments, the cell is derived from induced pluripotent stem cell (iPS cell)-derived retinal cell.In some embodiments, the cell is derived from retinal organoid. method

[0091] Described herein are methods for stimulating the regeneration of retinal ganglion cells and / or inducing retinal regeneration in a subject.Also provided are methods for enhancing retinal regeneration, improving retinal neurogenesis, enhancing retinal regeneration, restoring vision, and treating retinal degenerative disease, injury, damage, or blindness.

[0092] Also provided herein is a method for inducing retinal regeneration, comprising administering to a subject a composition described herein. In some embodiments, the method is effective to increase the number of Müller glia-derived retinal ganglion cells, induce Müller glia (MG) cells to enter the mitotic cell cycle, and / or generate new retinal neurons, including the generation of new ganglion cells. In some embodiments of the method, the number of retinal neurons is increased by at least 25% relative to a baseline level or other reference amount representing an untreated retina. In other embodiments, the number of retinal neurons is increased by at least 40%. In some embodiments, the number of retinal neurons is increased by 10%, 20%, 50%, 100%, 150%, 200%, or more.

[0093] Optionally, the methods disclosed herein may utilize combination therapy compositions that include one or more, or two or more small molecule reprogramming enhancing agents.The agents can be administered sequentially or simultaneously with the nucleic acid molecules disclosed herein.In another embodiment, one or more protein / peptide or miR-based reprogramming enhancing agents can be incorporated into the nucleic acid molecules used in the methods disclosed herein.In some embodiments, the methods are carried out in the absence of such reprogramming enhancing agents.

[0094] 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 has 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. Administration and Dosage

[0095] The compositions and / or nucleic acid molecules disclosed herein are administered in any suitable manner, often with a pharma- ceutically acceptable carrier.In the context of the present invention, there are suitable methods for administering compositions, compounds, molecules, nucleic acids, and vectors to the eye or retina of a subject, and a particular composition can be administered using more than one route, but in many cases, a particular route can provide a more rapid and effective response than another route.For the treatment of the retina, intraocular injection, such as but not limited to intravitreal injection and subretinal injection, is the most common delivery route to the retina.However, in some embodiments, periocular administration, suprachoroidal administration, systemic administration, or local administration is more suitable for delivery efficacy and safety.

[0096] The dose administered to a patient should be sufficient to induce a beneficial therapeutic response in the patient over time in the context disclosed herein.Therefore, the composition is administered to a subject in an amount sufficient to elicit an effective response and / or to alleviate, reduce, cure or at least partially prevent the symptoms and / or complications of retinal disease or injury.The appropriate amount to achieve this is defined as a "therapeutically effective dose".

[0097] The route, sequence and / or frequency of administration of the therapeutic compositions disclosed herein, as well as the dosage, may vary between individuals and may be readily established using standard techniques. In general, an appropriate dosage and treatment regimen provides the active compound(s) in an amount sufficient to produce a therapeutic and / or prophylactic effect. Such response may be monitored by establishing an improvement in clinical outcome in treated patients compared to untreated patients. EXAMPLES

[0098] The following examples are presented to illustrate the present invention and to assist one of ordinary skill in making and using the same, and are not intended to otherwise limit the scope of the invention in any way. Example 1: Reprogramming Müller glia to regenerate ganglion-like cells using developmental transcription factors

[0099] Many neurodegenerative diseases cause the degeneration of specific types of neurons. For example, glaucoma leads to the death of retinal ganglion cells, leaving other neurons intact. Neurons do not regenerate in the adult mammalian central nervous system. However, in non-mammalian vertebrates, glial cells naturally reprogram into neural precursors to replace neurons after injury. We have recently developed a strategy to stimulate the regeneration of functional neurons in the adult mouse retina by overexpressing the proneural factor Ascl1 in Müller glia. In this example, we test additional transcription factors (TFs) for their ability to direct regeneration to specific types of retinal neurons. Mice were engineered to express different combinations of TFs in MG, including Ascl1, Pou4f2, Islet1, and Atoh1. Using IHC, scRNA-seq, scATAC-seq, and electrophysiology, we find that retinal ganglion-like cells can be regenerated within damaged adult mouse retina in vivo by targeted overexpression of developmental RGC transcription factors.

[0100] In the past few years, our group and others have discovered strategies to stimulate adult mouse MGs to behave and generate neurons similar to their fish counterparts (4-9). Several transcription factors (TFs) differentially expressed between mouse MGs and retinal precursors were screened for their ability to stimulate MG neurogenesis in vitro (10). One factor that emerged from this screen was the proneural basic helix-loop-helix (bHLH) TF Ascl1, which can induce neurogenesis from MGs in vivo when co-treated with histone deacetylase [trichostatin A (TSA)] in young mice (11) and in adult mice (4). Ascl1-expressing MGs adopt a molecular phenotype similar to developing retinal precursors, and a subset of these cells undergo mitosis (5, 12). Some of these newly generated cells continue to differentiate into retinal neurons that connect to intrinsic circuits (4, 5). When Ascl1 is used to stimulate MG neurogenesis, most MG-derived neurons adopt a bipolar cell fate, with a minority resembling amacrine cells (4, 12). Recently, we reported that the efficiency and range of neural cell types generated by MG reprogramming can be significantly improved by adding the toneless class of bHLH TFs (Atoh1 / 7). With this combination, up to 80% of MGs expressing Ascl1 and Atoh1 become neurogenic precursors and ultimately neurons (13).

[0101] Our previous results show that combining two TFs of the same type (bHLH proneural) significantly increases the efficiency of MG reprogramming in vivo, although this paradigm did not allow control over the type of neurons generated by MGs. This is somewhat similar to the process of zebrafish regeneration, 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, certain retinal diseases are often the result of defects in specific neuronal classes. For example, in glaucoma, blindness results from the loss of retinal ganglion cells (RGCs) (16). Thus, there is an impetus to direct endogenous regeneration to specific diseased types of neurons for cell replacement strategies.

[0102] Herein, we test whether combining Ascl1 with other types of developmentally important TFs can more precisely direct MG-derived retinal precursors to specific retinal cell fates. Specifically, we investigate the effect of combining Ascl1 overexpression with two other TFs from different classes: the LIN, Islet1, MEC3 (LIM) homeodomain TF and the Pou4f2, class IV, Pit-Oct-Unc (POU) homeodomain TF. Both factors have been shown to be important for cell fate decisions in the developing retina, with Pou4f2 being required for RGC differentiation and Islet1 being important for the development of several types of retinal neurons, including RGCs (17, 18).

[0103] We find that expression of Pou4f2 and Islet1 in adult mouse MG, together with Ascl1, directs a subset of MG-generated neurons to a cell fate similar to RGCs. MG-derived RGC-like neurons (i) can be immunolabeled with markers of normal RGCs; (ii) have a similar transcriptome to developing RGCs by single-cell RNA sequencing (scRNA-seq); (iii) have broader electrophysiological properties, such as action potentials, than neurons generated by Ascl1 alone; and (iv) show a pattern of chromatin accessibility similar to developing RGCs. Taken together, our results indicate that combinations of developmentally relevant TFs can be used to direct neural regeneration from MGs to specific cell types.

[0104] Materials and Methods Primary cell culture Retinas from both sexes, tetO-Ascl1-GFP or tetO-Pou4f2-Islet1-Ascl1-GFP mice, were harvested for MG culture at postnatal day 12 (P12). To dissociate, retinas were incubated in papain and deoxyribonuclease (DNase) (Worthington) solution for 10 min at 37°C, followed by trituration. To stop the reaction, an equal volume of ovomucoid (Worthington) was added. Cells were then centrifuged at 300g for 10 min at 4°C and resuspended in growth medium consisting of Neurobasal (Gibco), 10% fetal bovine serum (FBS) (Clontech), N2 (Invitrogen), 1 mM L-glutamine (Invitrogen), 1% penicillin-streptomycin (Invitrogen), and mouse epidermal growth factor (100 ng / ml) (R&D Systems). Cells were seeded in 6-well dishes at a density of two retinas per 10 cm2 and incubated at 37°C. Medium was changed every 2 days until confluence was reached (approximately 7 days). At confluence, cells were passaged in 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 24 hours for at least 1 day. After thawing, cells were grown in FBS-reduced growth medium (1%) and treated with doxycycline to induce tetO-mediated genes.

[0105] animal All animals were treated and housed according to University of Washington Institutional Animal Care and Use Committee approved protocols. (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:tetO-P7I:tetO-Ascl1-ires-GFP mice were from a mixed background of C57BL / 6 and B6SJF1. Glast-CreER, LNLtTA, and rtTa mice were from Jackson Laboratory. tetO-mAscl1-GFP mice were provided by M. Nakafuku (University of Cincinnati), tetO-Atoh1 mice were provided by P. Chen (Emory University), and tetO-P&I mice were provided by X. Mu (University of Buffalo). Both males and females were used in the experiments with equal frequency. All in vivo experiments were performed in 40-day-old adult mice.

[0106] immunohistochemistry After CO2-mediated euthanasia, the corneas were removed and the eyes were fixed in 4% paraformaldehyde (PFA) in phosphate-buffered saline (PBS) for 30 min. The fixed eyes were then incubated overnight in 30% sucrose at 4 °C. The retinas were then frozen in optimal cutting temperature compound (OTC) and cryosectioned at 18 μm. For immunostaining, the sections were washed twice in PBS for 10 min and then incubated overnight in primary antibodies in 0.5% TritonX-10 and PBS. The slides were then washed in PBS and incubated in secondary antibodies for 1 h. Finally, the slides were washed again in PBS and coverslipped with Fluoromount-G (SouthernBiotech). See Table 1 for antibody and concentration information.

[0107] Cultured MG were plated on glass coverslips using FBS-reduced growth medium (as above). To activate the transgene, doxycycline was added at a concentration of 3 μg / ml every 24 h for 5 days. Coverslips were washed with PBS and then fixed with 4% PFA for 10 min at room temperature (RT), followed by 3 × 5 min washes with PBS. Primary antibody incubation was performed overnight at 4 °C in 0.5% TritonX-10 and 10% normal horse serum (NHS) in PBS. Coverslips were washed with PBS and then EdU labeled using click chemistry (Click-iT EdU Assay, Invitrogen), followed by incubation with secondary antibody and 4',6-diamidino-2-phenylindole for 1 h at RT. After staining, coverslips were washed three more times and mounted on slides using Fluoromount-G (SouthernBiotech). [Table 1] TIFF2025511192000003.tif103159

[0108] Fluorescence-activated cell sorting After euthanasia, retinas were dissociated into single cells as described for cell culture; after pelleting 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 FACSAriaIII cell sorter (BD Bioscience).

[0109] injection Intravitreal injections were performed using a 32G Hamilton syringe in mice anesthetized with isoflurane. NMDA injections were performed in a volume of 1 μl at a concentration of 100 mM in PBS. TSA (Sigma-Aldrich) was administered by intravitreal injection at a concentration of 1 μg / μl in DMSO. Intraperitoneal injections of tamoxifen (1.5 mg per 100 μl of corn oil) were administered to adult mice for 4 consecutive days to induce expression of the tetO-mAscl1-ires-GFP, tetO-P&I, and tetO-Atoh1 genes.

[0110] Microscopy / cell count Images were acquired with a Zeiss LSM880 confocal microscope. To quantify cell numbers, a minimum of four images were acquired per retina at the same magnification with a 20x objective.

[0111] Electrophysiology Recordings were performed identically to previous reports (4, 5). Mice were dark adapted before recording. After euthanasia, retinas were sliced ​​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 subject to recordings using infrared light and video differential interference contrast by confocal microscopy. Photoresponses were measured under infrared conditions and tissues were exposed to full-field illumination via blue and green light-emitting diodes. Recordings were performed using removed glass pipettes filled with a solution containing: 123 mM K-aspartate, 10 mM Hepes, 1 mM MgCl2, 10 mM KCl, 1 mM CaCl2, 2 mM EGTA, 0.5 mM tris-guanosine triphosphate, 4 mM MG-adenosine triphosphate, and 0.1 mM Alex aFluor695 hydrazide.

[0112] Construction of single-cell RNA libraries For the in vivo dataset, after FACS purification of GFP+MG, cells were centrifuged at 300g at 4°C and resuspended at a concentration of 1000 cells / μl. Library construction was performed using 10× Genomics 3' Single Cell RNA V3 or V3.1 Sequencing Kits as described by the manufacturer. Cells were encapsulated in gel beads with predefined unique barcodes using 10× Chromium Controller and 10× Genomics Chip Type G. Libraries were multiplexed using Single and Dual Index Kits (10× Genomics).

[0113] For in vitro experiments, MG cultures from 3–5 mice were used. After 5 days of doxycycline treatment, cells were dissociated using a mixture of Accutase (Sigma-Aldrich, Saint-Louis, MO) and DNAse (Worhington, Lakewood, NJ) for 2 min and then sedimented at 400 relative centrifugal force for 7 min at 4 °C. The pellet was resuspended in medium to reach a target concentration of 1000 cells / μl. Cells were passed through a strainer and loaded onto a 10xGenomics Chromium Single Cell chip G according to the protocol of the Chromium Single Cell 3' Reagent Kit v3.1 (10xGenomics, Pleasanton, CA). Single-cell RNA sequencing, mapping, and data analysis

[0114] Libraries were sequenced using an Illumina NextSeq500, 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 (cells identified as having low read depth or high mitochondrial content; >10%) were removed from the dataset. Microglia, and any astrocytes or endothelial cells present (for in vitro experiments) were identified and removed from the data prior to downstream analysis. Additionally, prior to analysis, gene expression data were normalized and scaled, and cells were clustered using principal component analysis and UMAP, using tools available in the Seurat R package version ≥3.0. Comparisons between data sets were performed by canonical correlation analysis as described by the Satija laboratory vignette (46) (https: / / satijalab.org / seurat / archive / v3.0 / integration.html).

[0115] Integration with developmental data To perform comparisons with the developing retina, data were first downloaded from Gene Expression Omnibus (GEO) (21). Label transfer was performed in Seurat using a reference dataset consisting of 432 randomly sampled cells of each major cell class (rod, cone, bipolar, amacrine, horizontal, retinal ganglion, and Müller glia) in the developing retina dataset, totaling 3024 cells identified by canonical markers. Reads were downsampled to a common average depth before analysis. Additionally, IPA neurons were directly merged with a subset consisting of only E14 cells from the development dataset.

[0116] Single-cell ATAC sequencingThe data obtained from sequencing was preprocessed using the Cellanger ATAC pipeline (2.0.0) (47). First, BCL files were converted to fastq and demultiplexed reads using “cellanger-atac mkfastq”. Then, “cellranger-atac count” was run to map Tn5 sites to mm10 (mouse genome), remove duplicated reads, and remove background cells. This returned peaks by Signac (48), a cell matrix and barcoded fragment files loaded into the R (4.0.4) (R core team, 2021) package. Macs2 was then run on the Signac object to barcode the fragment files and call peaks using Signac’s “Call Pealk” 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 that were outliers in 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 dimensions highly correlated with read depth; these LSI dimensions 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 various resolutions. Clusters were assigned to known retinal cell types by examining Tn5 insertions within 100 kb of known marker genes using Signac’s “CoveragePlot” and further corroborated using chromVAR scores of known lineage-specific TFs. Clusters of the same type were grouped together for visualization purposes. Vertebrate motifs were obtained from the Jaspar2020 database.These motifs were mapped to peaks in the Signac object using the Signac "AddMotifs" function. The Signac "RunChromvar" function was used to calculate the accessibility z-score of the motifs across all cells.

[0117] Integration of Data Sets Before integrating Signac objects, we first performed all previous computational steps on each sample separately. We then used BEDOPS(-m) (50) to create a shared peak set for all objects to be integrated. Signac's FeatureMatrix function was run on each sample, and the integrated peak set was used to put all samples into the same feature space. Samples were then downsampled to the same average read depth using the DropletUtils "downsampleMatrix" function. Samples were merged using Signac / Seurat's "merge" function, and standard Signac normalization, dimensionality reduction, clustering, and visualization were performed on the merged objects as described above. Objects were then split per sample, and samples were integrated with each other. Anchors between samples were calculated using "FindIntegrationAnchors", and then the integrated embedding space was calculated using Signac's "IntegrateEmbeddings" function. UMAP and clustering of integrated objects were performed as previously described.

[0118] Pseudo-Time Series Analysis To calculate pseudo-time series and identify trajectory branches, samples were loaded into Monocle3 (51) using the “as.cell_data_set” function of SeuratWrapper. Clustering was performed and partitions were defined using Monocle3 “cluster_cells”. Monocle3 “learn_graph” was then run to define the main trajectory graph. Pseudo-time series roots were placed into precursor-like or MG-like clusters using Monocle3 “order_cells”. Branches were selected using Monocle3 “choose_graph_segments”. Pseudo-time series and branching data were transferred to Seurat objects for further analysis.

[0119] Scatter Plot To generate scatter plots, the Signac / Seurat “FindMarkers” function was run between cell populations of interest using the top 25% of the most accessible peaks in those cell populations. A scatter plot was then generated showing the percentage of cells in each group that had accessibility to each peak. Peaks with a mean log base 2-fold change of >0.05 were selected for each group. For GO analysis, these peak sets were then loaded into GREAT (Genome Regions Enrichment of Annotations Tool) (52) or analyzed using the gprofiler2 package in R for scRNA-seq data.

[0120] Cascading Heatmap To construct the cascade heatmap, motif names were translated to mouse gene names using R and the “getLDS” function in biomart. The pseudo-time series lineage branches of RGCs were subset from the RNA and ATAC data of E14 samples. Variable motifs were identified by running the “addGCBias”, “getBackgroundPeaks”, “computeDeviations”, and “computeVariability” functions of chromVAR. Peaks with a linguistic score above 1.2 were retained for further analysis. This motif list was then subset again. Only motifs corresponding to TFs expressed in more than 10% of the cells in the RGC branch were retained. Finally, bound motifs were dropped. The motif enrichment scores of selected motifs were ordered across the pseudo-time series of E14 RGC branches of scATAC-seq data by fitting their ChomVAR scores to a third-order polynomial function and ordering the motifs by the maximum value of this function within our pseudo-time series range. To generate heatmaps, the accessibility z-scores of chromVAR motifs were plotted in the order previously derived across pseudotime within the RGC lineage. RNA heatmaps were generated by plotting genes whose binding sites are in the final motif list across pseudotime in the same order across RNA objects within the RGC lineage. Cascade heatmaps of reprogrammed cells were generated by plotting the motifs and factors identified in the developmental data.

[0121] result Pou4f2 and Islet1 increase the diversity of neurons from Ascl1-reprogrammed MG

[0122] We previously developed mice in which Ascl1 was specifically induced in 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) induced MG to express genes related to developing retinal precursors and generate new neurons in vivo in the adult mouse retina. Most of these newly generated neurons adopt a bipolar cell fate (4, 5, 12). To examine whether expression of additional TFs (Pou4f2 or Islet1) could direct MG-derived precursors to other neuronal fates, Glast-CreER:LNL-tTA:teto-mAscl1-GFP mice were crossed with a 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, this cassette allows expression of either Pou4f2, Islet1, or possibly both.

[0123] The tetO-IPA mouse line allows us to test whether MG reprogramming is enhanced by treatment with Pou4f2+Ascl1, Islet1+Ascl1, or Islet1+Pou4f2+Ascl1 (hereafter IPA). After intraperitoneal application of tamoxifen to induce TFs in MG, retinal damage was induced by intravitreal injection of NMDA followed by TSA (Figure 1B). Mice were then euthanized 3 weeks later for immunofluorescence analysis to evaluate the fate of recombinant MG (Figure 1, C–E). Figure 1 (C and D) shows that this protocol induces transgenes in MG and neurons derived from them [green fluorescent protein positive (GFP+)]. We found GFP+ MG-derived cells expressing either Brn3 (Pou4f2) (17%), Islet1 (33%), or both TFs (7%) (Figure 1, C and D).

[0124] Further immunohistochemistry (IHC) analysis showed that the combination of IPA effectively promoted MG-induced neurogenesis. Most glial-derived cells acquire 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 ganglionic / amacrine markers HuC / D (Figure 1, F and G) or bipolar marker Otx2 (Figure 1, G and H). Expression of IPA significantly enhanced MG neurogenesis of HuC / D neurons compared with Ascl1 alone (Figure 1G). Consistent with previous reports, a subset of MG-derived neurons was derived from proliferative MG, EdU+ (Figure 8A-8C). Taken together, these data suggest that the addition of TFs Pou4f2 and / or Islet1 enhances the neurogenic capacity and expands the cell fate of the resulting Ascl1-MG in vivo.

[0125] We previously found that the combination of Ascl1:Atoh1 could stimulate neurogenesis from MG in the absence of retinal injury (13). In contrast, IPA induction in uninjured retina was not sufficient to stimulate neurogenesis (Figure 9, A, B, and E). We also tested whether MG undergoes neurogenesis reprogramming when we activate IPA factors in MG after NMDA injury and TSA, but not before NMDA and TSA, as shown in Figure 1. We found that induction of IPA after injury stimulated a small amount of neurogenesis, but much less than in our original paradigm (Figure 9, C-E).

[0126] Because a significant portion of newly generated neurons in the injured retina expressed the ganglionic / amacrine 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 wide-field amacrine cells, and cells with this morphology have not been seen previously in Ascl1 alone (Figure 2, A-F).

[0127] IPA induces neurons with an RGC-like transcriptome We next 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 the cell fate changes induced by IPA to those caused by expression of Ascl1 alone, we used Seurat to merge data from IPA treatment with a previously acquired Ascl1-only reprogramming library (4) and cluster cells (Figure 3A). The combined data from the IPA experiment and the prior Ascl1 dataset were projected onto a single uniform manifold approximation and projection (UMAP) plot, and clusters of cell types were identified by known marker genes (Figure 3B).

[0128] The UMAP plot of Ascl1 alone combined with IPA treatment includes clusters of cell types previously observed during Ascl1-mediated reprogramming (e.g., MG, precursors, and bipolar cells) (Figure 3A). This analysis revealed two additional phenotypes unique to the IPA condition. First, the neurogenic efficiency of MG was increased by IPA 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 potently stimulates neurogenesis from adult MG. Second, after IPA treatment, novel clusters appeared that were not present in the Ascl1 alone condition (Figure 3C). Cells expressing Islet1 but not Pou4f2 were found primarily in bipolar clusters, whereas cells expressing Pou4f2 were found primarily in novel RGC-like clusters, with or without detectable Islet1 (Figures 10A-10C).

[0129] The new cell cluster induced by IPA shows high expression of genes characteristic of RGCs, such as Elavl4 and Sox11 (Figure 3B) (20). Furthermore, we found that these IPA neurons express many genes found in the Gene Ontology (GO) terms "axon guidance," "axon elongation," and "axon formation" (Figure S10D). Because Islet1 and Pou4f2 are upstream of the regulatory network that induces RGC fate, we assayed whether this combination of factors could induce multiple RGC genes in this cluster of MG-derived neurons. Using Seurat's label transfer function, we broadly compared the transcriptome of IPA neurons to reference datasets of all major retinal neuron classes (21, 22). The new cluster was classified as an RGC cluster (average prediction score = 0.74), suggesting an overall transcriptome similarity to native RGCs (Figure S10E). Compared to Ascl1-induced neurons, a significant number of RGC-associated genes were expressed in IPA-induced neurons (Figure 3D). These include genes such as Sox4 and Sox11, which are redundantly required for the acquisition of RGC fate (23, 24), and the axon growth-related gene Gap43, which is highly expressed in developing RGCs (25). Both Satb1 and Cntn5 were found to be expressed in a subset of IPA-induced neurons. Satb1 is highly expressed in the on-off direction-selective subtype of RGCs, and Satb1 controls the expression of Cntn5 (26). Using IHC, we were able to confirm that IPA-induced neurons expressed Satb1 protein (Figure 3E). In addition, the RGC and amacrine marker calretinin (Calb2) was also detected at the RNA and protein levels (Figure 3, C and F).

[0130] Despite this large set of RGC genes expressed by MG-derived RGC-like neurons, these cells fail to express some canonical RGC markers, such as Pou4f1, Sncg, Rbpms, and Nefm (Figure 3D). This suggests that the newborn neurons are not fully differentiated into mature RGCs. Consistent with this observation, when we integrate the scRNA-seq data of IPA-induced neurons with a dataset from E14 (embryonic day 14) embryonic mouse retina (21), we find that MG-derived RGC-like neurons most closely resemble immature RGCs (Figure 3G). When we compared IPA-induced neurons from a 3-week endpoint with those with a longer survival period (6 weeks), we found that the regenerated RGC-like neurons were a stable population and did not show evidence of increased cell death or stress during this period (Figures 11A-D).

[0131] Co-expression of Islet1-Pou4f2 and Ascl1 induces a more uniform RGC state from MGs in vitro Because the tetO-Pou4f2 and Islet1 transgenic constructs contain mutually exclusive loxP sites, most of the MGs expressing Cre in vivo expressed Ascl1 and either Pou4f2 or Islet1. Although a small number of cells were colabeled for both Pou4f2 and Islet1 in vivo (Figure 1, C and D), to better evaluate the effect of uniformly overexpressing all three transgenes, mice containing tetO-Pou4f2-Islet1 and tetO-Ascl1-GFP cassettes were injected into the germline Rosa26-rtTA line and bred, and then MG reprogramming experiments were performed in vitro (Figure 4, A and B). MGs were cultured from postnatal day 1111 mice for 7 days and then passaged as previously described (10). Cultures obtained in this manner consisted mostly of MGs, although some surviving neurons were observed (10). 5-ethynyl-2'-deoxyuridine (EdU) was added to the medium to determine which cells were derived from proliferative MG and which were likely surviving neurons from the initial dissociation. After passaging, doxycycline was added to the medium to induce transgene expression, and cells were then assayed by immunofluorescence and scRNA-seq (Figure 4B).

[0132] Analysis of the cultures 5 days after treatment confirmed that these cells expressed the transgene. Immunolabeling of the transgene showed that the cells co-expressed Pou4f2, Islet1, and Ascl1 (Figure 12A). These cells adopted a predominantly neuronal morphology, and immunolabeling showed EdU+ cells co-labeled with the neuronal markers Tuj1 (Figure 4C), neurofilament (Figure 4D), and calbindin (Figure 4E).

[0133] To determine how IPA overexpression differs from Ascl1 alone, we repeated the reprogramming experiment described above with sister MG cultures from tetO-Ascl1;Rosa26-rtTA mice with or without doxycycline treatment to activate transgene expression. For each sample, scRNA-seq was performed as described above. Untreated MG were largely homogenous, containing one glial cluster and a small cluster containing only a few surviving neurons (Figure S12, B and C). To compare cluster composition between treatment conditions and with the in vivo IPA dataset, all three were merged together (Figure 4, F-I, and Figure S12D). Cells that mapped to neuronal clusters from the IPA in vivo dataset were identified in both Ascl1-only in vitro and in vitro reprogrammed with IPA. However, each treatment stimulated one major population of neurons from the MG; in Ascl1 cultures, neurons most closely 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 differentiating into bipolar cells. This differed from our in vivo observations, where the bipolar clusters were proportionally similar to those in Ascl1-only samples (Figs. 1D and 4, F and H).

[0134] 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 primarily restricted to the Ascl1-only neuronal cluster. RGC genes, such as Grin2a and Calb1, were found exclusively in IPA neurons (Figure 4J). To identify unique marker genes expressed in IPA neurons, we created subsets of the total neuronal population in the IPA and Ascl1-only combined datasets (Figure 4K) and performed differential gene expression (DGE) analysis (Figure 4L). We identified a number of RGC genes enriched in IPA neurons, whereas bipolar genes were enriched in Ascl1 neurons. In addition, we also found several canonical markers of RGC lineages, such as Sncg, Nefm, and Pou4f1, which were induced in vitro but not in vivo (Figure 4M). We also failed to detect any cells expressing the endogenous light-sensitive RGC marker Opn4 (Figure S12E). Taken together, these results suggest that simultaneous overexpression of all three IPA factors biases MG-derived neurons toward an RGC-like fate and induces more uniform RGC gene expression.

[0135] IPA induces neurons with diverse electrical properties We previously found that Ascl1 can stimulate MG-derived neurons with physiological properties of intrinsic retinal neurons, especially bipolar cells (4, 13). Because IPA treatment results in a distinct molecular and morphological neuronal phenotype compared to Ascl1 alone, we characterized the light response and electrical properties of these cells. Patch clamp electrophysiology was performed on GFP+ cells in retinal slices and whole mounts after the IPA in vivo regeneration paradigm. Responses to current and voltage steps, as well as responses to light stimulation, were measured. The membrane resistance and resting potential of IPA-induced neurons compared to GFP-MG, intrinsic neurons, and neurons from previous regeneration strategies were plotted using Ascl1 or Ascl1:Atoh1 (Figure 5A) (4, 5, 13). Glial cells have low membrane resistance, hyperpolarized resting potentials, and little voltage-activated conductance. Neurons have higher resistance, less negative resting potentials, and express voltage-activated conductance.

[0136] Most of the IPA-induced neurons showed resting potential and membrane resistance profiles similar to endogenous neurons; however, some of these cells still had glial-like hyperpolarized membrane potentials (Figure 5A). This is consistent with our IHC and scRNA-seq analysis, where a portion of the glia does not reprogram after IPA treatment (see Figures 1 and 3). Figure 5B shows examples of responses to families of current or voltage steps recorded from IPA-treated GFP+ cells. These cells exhibit a variety of characteristics. Some appear to have a neuronal phenotype and express a voltage-activated K+ conductance that limits the extent of depolarization to the current step, while others retain glial features (Figure 5B). Six of the 16 recorded cells responded to a brief light flash, indicating that they established synaptic connections with other components of the retinal circuit (Figure 5C). Some cells responded to the current step by generating action potentials, while others generated spiking activity likely representing Ca2+ spikes (Figure 5D). This diversity of physiological properties is consistent with the phenotypes observed in Figures 1 and 3. Notably, previous strategies to stimulate MG-derived neurons did not observe action potentials, a defining feature of RGCs (4, 5, 13). Thus, IPA increases the diversity of electrical properties of MG-derived neurons, including generating some cells capable of generating Na+ and / or Ca2+ action potentials.

[0137] Expression of IPA remodels MG chromatin into an incomplete RGC-like fate Next, to better understand the mechanistic differences between these two reprogramming strategies, we performed single-cell assays for transposase-accessible chromatin sequencing (scATAC-seq) in Ascl1 vs. IPA reprogramming MG. Mice were treated with the same in vivo retinal regeneration paradigm described in Figure 3, but for scATAC-seq rather than scRNA-seq. Nuclei (1692) from Ascl1 only and 2451 from IPA treatment passed our quality control criteria (see Materials and Methods). Single cells from these two treatments were then merged and plotted as UMAPs to identify cell types (Figure 6, A-C). Cell type clusters were identified by the patterns of accessible chromatin-proximal genes identified in specific retinal cell types. The coverage plot shows representative peaks for the groups we identified: MG (Rlbp1+), neurogenic transition (Islet1+), MG-derived bipolar cells (Crx+), induced RGC-like cells (Pou4f2+), and photoreceptors (Arr3+) (Figure 6D). Dividing the UMAP between the two treatment groups reveals that both Ascl1 and IPA conditions result in MG-derived bipolar neurons, while large cell clusters retain the MG phenotype (Figure 6, B and C). However, IPA treatment induces a unique cell cluster with accessible chromatin that most closely resembles RGCs (Figure 6C). Consistent with the accessibility pattern, motifs for Otx2 are enriched in the bipolar cluster, whereas Pou4f2 motifs are well represented in the accessible chromatin of the IPA-specific cluster (Figure 6F). This is consistent with our scRNA-seq findings, demonstrating that these MG-derived neurons have a pattern of cis-regulatory regions consistent with their transcriptomic identity.

[0138] To determine the extent to which the chromatin of MG-derived RGCs is similar to that of normal developing RGCs, we generated scATAC libraries from E14 developing mouse retina and compared them to MG-derived RGCs (Figure 13A-13F). The scatter plot in Figure 6G shows that there is a significant correlation between the accessible peaks of E14 RGCs and MG-derived neurons, however, there are clearly many regions that are differentially accessible between cell types (Figure 6G). When we assayed for GO enrichment to understand the types of genes that are more highly represented in nearby accessible chromatin in E14 RGCs than in MG-derived RGCs, many of the top terms were related to axon growth (Figure 6H and Table 2). This is consistent with the fact that many MG-derived RGCs do not extend long axons.

[0139] Current models of retinal development suggest that a cascade of TFs is activated sequentially as cells transition through multipotent precursors to neurogenic precursors to their final neuronal fate (27). Therefore, we sought to investigate whether MG-induced neurogenesis recapitulates the developmental cascade at the chromatin level. Using pseudotime series, we sequenced cells in the putative lineage from progenitors to RGCs in E14 retina and compared this to a similar analysis of MG-derived RGCs, and then analyzed the motif enrichment of accessible chromatin in a pseudotime series of “cascade plots” (Figure 6, I and J) (28). This analysis reveals important differences in the process of RGC development and RGC production from MGs. Normal development of RGCs shows a downregulation of precursor TF motifs (Rax and Sox2) in accessible regions, followed by a transient increase in regions with a bHLH motif (Atoh7), followed finally by the induction of regions with mature RGCTF motifs (Pou4f1 / 2 / 3, Ebf1, and Onecut). This has been recently described in both mice and humans (28, 29). In contrast, the sequential changes in motif representation in accessible chromatin in MGs reprogrammed to generate neurons with IPA show clear differences from normal development. Although motifs of precursor genes are reduced as cells acquire an RGC-like identity, precursor TFs are apparently never completely downregulated as their motifs persist in accessible regions of MG-derived RGCs (Figure 6J). Furthermore, MG-derived RGCs show an increase in accessible bHLH motifs and a robust increase in Pou4f1 / 2 / 3 motif expression in accessible chromatin, although putative downstream TFs of Pou4f1 / 3, such as Onecut and Ebf, are not sequentially activated (Figure 6J). This epigenomic analysis suggests that RGC-like generation from IPA-treated MG is incomplete, in part due to the maintenance of accessibility in glial and progenitor regulatory regions. Consistent with this notion, the progenitor / MG marker Sox2 is still detectable by immunofluorescence 3 weeks after IPA reprogramming in GFP+ cells with neuronal morphology (Figure 6K).

[0140] Atoh1 can improve the ability of IPA to induce MG-derived RGC-like cells scRNA and scATAC-seq analysis revealed that IPA-induced neurons resemble RGCs, but these cells lack some characteristics of mature RGCs. One hypothesis as to why this is the case is the persistence and chromatin accessibility of glial and progenitor genes in MG-derived neurons (i.e., Sox, Rax, Vsx2, etc.). Recently, we reported that the Atoh class of TFs can potently stimulate neurogenesis when combined with Ascl1 (13). The combination of Ascl1:Atoh1 results in approximately 80% of transgene-expressing MGs acquiring neural identity. Therefore, we tested whether combining Atoh1 with IPA reprogramming could improve the regeneration of RGCs from MGs in vivo.

[0141] Mice containing tetracycline-inducible Atoh1 were crossed with the IPA strain and the regeneration paradigm was performed as described in Figure 1. We then performed scRNA-seq and IHC analysis of MG progeny as described above (Figure 7, A and B). Consistent with previous findings using Atoh1, expression of IPA and Atoh1 in MG caused the majority of MG progeny to acquire neuronal identity (13). IHC revealed that most GFP+ MG-derived neurons expressed the ganglionic / amacrine markers HuC / D and lacked expression of the bipolar marker Otx2 (Figure 7C). Adding Atoh1 to the IPA factor combination also circumvents the requirement of retinal injury to induce MG neurogenesis (Figures 14A-14D). We then performed scRNA-seq on regenerated cells treated from the IPA:Atoh1 condition and merged them with cells from the IPA-alone treatment group to determine whether the addition of Atoh1 could improve RGC generation after retinal injury (Figure 7D). This analysis revealed that MG-derived neurons from IPA:Atoh1 mice were most similar to MG-derived RGCs from IPA conditions, but also contained low proportions of bipolar, pyramidal, and amacrine cells (Figure 7D, and Figures S14E and S14F).

[0142] To assess whether Atoh1 overexpression reduces the progenitor signature of MG-derived neurons after IPA expression, a subset of RGC-like cells from each dataset was formed for comparison. DGE analysis revealed that in IPA:Atoh1 conditions, RGC-like cells showed a greater reduction in expression of progenitor and glial genes than similar cells in IPA-only conditions (Figure 7E). This was further confirmed by GO analysis, where IPA:Atoh1 reduced genes associated with neural progenitors compared to IPA alone conditions (Figure 7F), consistent with the hypothesis that Atoh1 promotes maturation of RGC-like cells.

[0143] We previously showed that Ascl1:Atoh1 generates immature RGC-like cells from MG (13). We hypothesized that adding IPA factors to Atoh1 might induce markers of more mature RGCs. We compared IPA:Atoh1 to our previously generated Ascl1:Atoh1 dataset and found a similar composition of cell types (Figure 7G and Figures S14E-S14H). However, we found that addition of IPA to Atoh1 led to a 15% increase in RGC-like cells and a corresponding decrease in bipolar and progenitor-like cells (Figures S14G and S14H). DGE analysis revealed that addition of IPA to Ascl1:Atoh1 led to enrichment of RGC genes, such as Map2 and Thy1 (Figure 7H). Furthermore, the top GO terms enriched in IPA:Atoh1 were related to neuronal process development and axon formation (Figure 7I), and many genes were found to be specifically related to axon guidance (e.g., Epha8 and Sema3e) and synaptogenesis (e.g., Syt1 and Snap25). [Table 2] TIFF2025511192000005.tif33159

[0144] Consideration Non-mammalian adult vertebrates can regenerate neurons in many regions of the central nervous system (CNS). For example, after tail amputation in larval frogs and some adult urodeles, radial glial cells in the spinal cord acquire a pattern of gene expression similar to neuronal precursors and continue to proliferate and regenerate an apparently normal spinal cord (30). Similarly, in the zebrafish retina and brain, glia respond to injury by activating a precursor-like gene expression program of TFs (31). These glial-derived progenitor cells undergo multiple rounds of mitotic cell divisions, and progeny differentiate into a range of neuronal types that can restore brain and retinal function (32).

[0145] Re-expression of developmentally active TFs in adult mammalian glia triggers a regenerative process in these cells that is similar in many ways to that seen in fish and amphibians. For example, after retinal injury, transgenic overexpression of proneural TF Ascl1, in combination with histone deacetylase inhibition, can stimulate MGs to acquire a progenitor-like state with the capacity to generate bipolar neurons (4). MG-derived neurons differentiate in that they form synapses with surrounding neuronal circuits and respond to light. In addition to systematically tracing neurons to validate their glial origin, we used EdU labeling to show their adult origin and profiled cells using scRNA-seq and scATAC-seq to observe intermediate states between glial precursors and regenerating neurons (5, 12, 13). Taken together, this validates that TFs can reprogram adult CNS glia to generate neurons.

[0146] Although Ascl1 induces MGs to adopt many characteristics of retinal progenitor cells, such as proliferative neurogenesis, and transcriptional and epigenetic landscapes similar to those of developmental progenitors, not all developmentally appropriate Ascl1 targets are induced in MG-derived progenitors, and neuronal output from Ascl1 MGs is restricted primarily to bipolar neurons (5). Thus, we reasoned that additional TFs may be required to properly induce MG-derived precursors into specific types of neurons. For example, glaucoma is primarily caused by the death of RGCs.

[0147] RGCs are generated during development by a cascade of TFs characterized by the early expression of Atoh7 and the downstream expression of additional TFs such as Pou4f1 / 2 and Islet1 (33). Atoh7 is required for proper RGC fate by inducing these downstream stabilizing TFs (34-36). Two of these downstream TFs, Pou4f2 and Islet1, are required for proper RGC fate specification (18, 37, 38), and ectopic expression of Pou4f2 and Islet1 in Atoh7 null retinas is sufficient to rescue RGC fate (19). Taking advantage of our extensive knowledge of the normal transcriptional regulation of RGC fate, we were able to test whether members of this TF cascade could reinitiate the generation of these cells from Ascl1-induced MG-derived precursors.

[0148] Here, we report that the RGC fate inducers Islet1 and Pou4f2, together with Ascl1, can induce MG neurogenesis towards an RGC-like fate. Using IHC, we show that MG-derived neurons derived from IPA-expressing MG express protein markers and morphological characteristics of RGCs. At the physiological level, these IPA-induced MG-derived neurons display neuronal resting membrane potentials and a subset of generated voltage-dependent action potentials, consistent with an RGC-like fate. Finally, our molecular analysis using scRNA-seq and scATAC-seq indicates that at the transcriptomic and epigenetic levels, IPA-induced MG-derived neurons most closely resemble immature RGC cells.

[0149] Although expression of IPA transgenes in MG collectively reprograms a subset of cells to an RGC-like fate, this combination does not activate the full complement of TFs required for mature RGCs. In both scRNA-seq and scATAC-seq datasets, we found that some mature RGC genes are not induced. Instead, MG-derived neurons retain expression of some precursor / glial genes, resulting in a chromatin landscape intermediate between precursors and RGCs. The progenitor state of MG can be suppressed by expression of Atoh1 in addition to IPA, consistent with a role for atonal TFs in promoting neuronal differentiation, which allows further differentiation of MG-derived RGCs. Taken together, our results show that ectopic expression of developmental TFs essential for a cell type-specific trajectory can reprogram MGs toward that same trajectory. This suggests a holistic approach in which the complementary and sequential actions 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 order would provide a more effective strategy for reprogramming.

[0150] Interestingly, RGC-like neurons derived from MG are most frequently found in the internal nuclear layer (INL) instead of the ganglion cell layer (GCL). Although a small percentage of RGCs is usually found in the INL in mice, most migrate to the GCL. It is possible that the inner plexiform layers are a barrier to migration of RGC-like cells, or alternatively, those layers may lack cues for proper migration. Although scRNA-seq data show that MG-derived RGC-like cells express many RGC genes, they may lack a critical migration program. Nevertheless, some of these cells respond to light by connecting with existing neural circuits, and it is possible that they can establish proper connectivity even in the absence of a suitable in vivo location.

[0151] Also, although we did not observe strong axonal growth directed toward the optic nerve, we found that many genes important for axonal growth and guidance are expressed in RGC-like cells. It is possible that some important guidance factors are not expressed in these cells, or alternatively, the adult retinal environment does not express the guidance factors necessary to direct axons toward the optic nerve head. RGC transplantation experiments showed 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.

[0152] Over the past decade, numerous reports have suggested that other developmental TFs, such as Sox2, Pax6, NeuroD1, Neurog2, and Ascl1, can stimulate neurogenesis from glia both in vitro and in the brain (39). However, the interpretation of many of these reports has been obscured by the discovery that lineage tracing of new neurons using existing adeno-associated virus (AAV) paradigms is unreliable, often leading to endogenous neurons being misidentified as glial in origin (40, 41). Nevertheless, some of the earlier studies using alternative methods to trace glial-derived neurons were able to show that astrocyte-to-neuron reprogramming is possible and that proneural TFs, including Ascl1, are among the neurogenesis stimulators (42-45). Thus, the strategy of combining TFs from specific nervous systems, as used in the retina, may have applicability throughout the CNS.

[0153] Overall, this study provides a proof of concept that TFs used to specify cell fates during development can be utilized to initiate similar fate acquisition from reprogrammed glial cells in the adult CNS. The process of reconstitution of cell fate acquisition during endogenous regeneration strategies is key to directing appropriate neural replacement in specific neurodegenerative diseases.

[0154] References 1.L.Todd,TAReh,Cold Spring Harb.Perspect.Biol.14,a040816(2022). 2. A. Hamon, et al. Dev. Dyn. 245, 727-738 (2016). 3.A.Bringmann,et al.Prog.Retin.EyeRes.28,423-451(2009). 4.NLJorstad,et al.Nature548,103-107(2017). 5.NLJorstad,et al.Cell Rep.30,2195-2208.e5(2020). 6.T.Hoang,et al.Science370,eabb8598(2020). 7.E.M.Rueda,et al.Cell Rep.27,1637-1649.e6(2019). 8.K.Yao,et al.Cell Rep.17,165-178(2016). 9.K.Yao,et al.Nature560,484-488(2018). 10.J.Pollak,et al.Development140,2619-2631(2013). 11.Y.Ueki,et al.Proc.Natl.Acad.Sci.U.S.A.112,13717-13722(2015). 12.L.Todd,et al.Cell Rep.33,108507(2020). 13.L.Todd,et al.Cell Rep.37,109857(2021). 14.C.Powell,et al.Sci.Rep.6,24851(2016). 15.F.D.D’Orazi,et al.J.Comp.Neurol.528,2816-2830(2020). 16.G.Beykin,et al.Prog.Retin.Eye Res.80,100875(2021). 17.Y.Elshatory,et al.J.Comp.Neurol.503,182-197(2007). 18.L.Gan,et al.Proc.Natl.Acad.Sci.U.S.A.93,3920-3925(1996). 19.F.Wu,et al.Proc.Natl.Acad.Sci.U.S.A.112,E1559-E1568(2015). 20.A.Usui,et al.Development140,740-750(2013). 21.B.S.Clark,et al.Neuron102,1111-1126.e5(2019). 22.T.Stuart,et al.Cell177,1888-1902.e21(2019). 23.KCChang,et al.J.Neurosci.37,4967-4981(2017). 24.Y.Jiang,et al.J.Biol.Chem.288,18429-18438(2013). 25.TAReh,et al.J.Neurobiol.24,949-958(1993). 26.Y.-R.Peng,et al.Neuron95,869-883e866(2017). 27.EABassett,et al.Trends Neurosci.35,565-573(2012). 28.C.Finkbeiner,et al.Cell Rep.38,110294(2022). 29.P.Lyu,et al.Cell Rep.37,109994(2021). 30.ADKakebeen,et al.eLife9,e52648(2020). 31.BWLindseyet al.Prog.Neurobiol.170,99-114(2018). 32.D.Goldman,Nat.Rev.Neurosci.15,431-442(2014). 33.F.Wu,et al.Nat.Commun.12,1465(2021). 34.NLBrown,et al.Development128,2497-2508(2001). 35.JNKay,et al.Neuron30,725-736(2001). 36.J.Brodie-Kommit,et al.Sci.Adv.7,eabe4983(2021). 37.X.Mu,et al.Proc.Natl.Acad.Sci.USA105,6942-6947(2008). 38.L.Pan,et al.Development135,1981-1990(2008). 39.R.Bocchi,et al.Neuron110,366-393(2022). 40.S.Blackshaw,et al.J.Clin.Invest.131,e146134(2021). 41.L.-L.Wang,et al.Cell184,5465-5481.e16(2021). 42.C.Lentini,et al.CellStemCell28,2104-2121.e10(2021). 43.O.Torper,et al.Proc.Natl.Acad.Sci.USA110,7038-7043(2013). 44.C.Heinrich,et al.Stem Cell Rep.3,1000-1014(2014). 45.N.Mattugini,et al.Neuron103,1086-1095.e5(2019). 46.A.Butler,et al.Nat.Biotechnol.36,411-420(2018). 47.ATSatpathy,et al.Nat.Biotechnol.37,925-936(2019). 48.T.Stuart,et al.Nat.Methods18,1333-1341(2021). 49.Y.Zhang,et al.Genome Biol.9,R137(2018). 50.S.Neph,et al.Bioinformatics28,1919-1920(2012). 51.J.Cao,et al.Nature566,496-502(2019). 52.CYMcLean,et al.Nat.Biotechnol.28,495-501(2010). 53.NMTran,et al.Neuron104,1039-1055.e12(2019).

[0155] Example 2: Promoting RGC production using transcription factors specific for developing retinal cells. To determine which transcription factors may help promote the production of retinal ganglion cells from Müller glia, we used lentiviruses to induce the expression of several transcription factors 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 (arrows in Figure 15) was able to induce Müller glia to generate new cells with gene expression characteristic of RGCs, including Nefm, Nefl, Ebf1 and Gap43 (Figure 16).

[0156] Example 3: AAV-delivered reprogramming transcription factors can induce neurogenesis. Figure 17A-17C. Shows the protocol used to demonstrate that the lineage of HuC / D+ neurons was traced from MG by tdTomato. Immunofluorescence markers indicate that successful delivery of reprogramming transcription factors resulted in reprogrammed HuC / D+ neurons from MG cells. As shown in Figure 18, both Atoh1 and Atoh7 delivered by AAV can induce HuC / D+ neurons. Atoh1 and Atoh7 are nearly equally efficient. AAV delivery of reprogramming factors under these conditions was much less efficient than transgenic expression.

[0157] We tested five additional RGC transcription factors: Irx2, Irx5, Neurod2, Ebf1, and Tcf3. Of these, Irx2 and Neurod2 promote axonal growth in IPA-reprogrammed MG (Figure 19). This indicates that certain RGC transcription factors can increase some specific RGC genes.

[0158] Example 4: Human Müller glia can be generated in vitro from fetal retina or pluripotent stem cells The protocol of embryonic stem cell (ESC) differentiation to generate retinal organoids (RO) is illustrated diagrammatically in Figure 20A. Images of MG development over time in retinal organoids labeled with RLBP1 and SOX2 are shown in Figure 20B. The upper panel of Figure 20C shows the schematic protocol for the generation of retinosphers (RS), and the lower panel shows images of retinosphers made from several fetal retinas and cultured for various times to be labeled. Characterization of MG in RS by RLBP1, VSX2, SOX9, SOX2, and GFAP is shown in Figure 20D.

[0159] Human MGs can be reprogrammed with ASCL1 to generate neurons in dissociated cultures (Figure 22). These neurons express several pan-neuronal markers, such as DCX and TUJ1, but are not mature. Human MGs can be derived from either retinal organoids or fetal human retina. Both glial sources respond similarly to ASCL1. Example 5: Promoters driving expression in Müller glia

[0160] Characterization of the ShH10 capsid and RLBP1 promoter in non-human primate (NHP) Müller glia is illustrated in Figure 23. We found that the human RLBP1 promoter works very well to drive expression in Müller glia (mouse, human or NHP), but it is 2.8 kB and too complex for 2-3 reprogramming TFs. Therefore, we started working with a much smaller promoter: HES1. The HES1 promoter provides a good alternative to the RLBP1 promoter. The HES1 promoter is only 337 bp and is expressed in adult Müller glia, as well as in retinal precursors. Furthermore, HES1 expression increases after ASCL1 infection. As shown in Figure 25, the HES1 promoter drives very good expression especially in Müller glia of human retinal organoids. GFP is expressed in HES1 and Sox9 positive cells 7 days after infection with the Hes1-GFP construct. This construct is specific at day 7. Figure 25C shows that GFP is not expressed in OTX2-positive cells (HES1-GF), but GFP / OTX2-positive cells are obtained after expression of ASCL1 (reprogramming cells, Figure 25D). The HES1 promoter also directs GFP expression in MG in adult NHP dissociated cultures, as shown in Figure 26.

[0161] This small specific promoter offers the possibility for a single AAV virus to drive reprogramming TFs in Müller glia. We have created the virus shown in Figure 24, along with more complex "autoregulatory loop" viruses to allow tracking of reprogrammed cells over time. The HES1 promoter shows specificity. The related HES5 provides another alternative.

[0162] Throughout this application, various publications are referenced. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this invention pertains.

[0163] 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 deviating from the spirit and scope of the invention. Accordingly, the invention is not 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, and Tcf3, and combinations 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 factor comprises 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 factor comprises Irx2 and / or Neurod2.

7. 7. The nucleic acid molecule of claim 1, 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 according to 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. 13. The composition of claim 12, wherein the HDAC inhibitor is trichostatin A (TSA), (Pro) / romidepsin, (Pro) / belinostat, (Pro) / panobinostat, and / or (Pro) / vorinostat.

14. 12. A method for stimulating regeneration of retinal ganglion cells in a subject, comprising administering to the subject's retina a composition according to claim 11, thereby stimulating regeneration of retinal ganglion cells.

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

16. 15. 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 at a subsequent time, a second administration 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 being treated for a retinal disease, injury, or degeneration.

18. 15. 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. 20. The method of claim 19, wherein the vector is a non-viral vector or a viral vector.

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

22. 15. The method of claim 14, wherein said administering to the retina is an intravitreal injection or a subretinal injection.

23. 16. 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 and / or second nucleic acid molecule is mRNA.

25. 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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