WO2026019376A1 - Reprograming of pluripotent stem cells into ganglion progenitors using a neuronal differentiation transcription factor for treatment of ocular disorders - Google Patents

Abstract

Disclosed is a method of producing ganglion progenitor cells from pluripotent stem cells and their use for treating ocular disorders. In particular, disclosed is a method of producing ganglion progenitor cells from pluripotent stem cells by transducing pluripotent stem cells with the neuronal differentiation transcription factor Neuronal Differentiation 1 (NEUROD1) in a chemically-defined and xenogenic-free culture medium and their use for treating ocular disease and/or an optic neuropathy.

Description

REPROGRAMING OF PLURIPOTENT STEM CELLS INTO GANGLION PROGENITORS USING A NEURONAL DIFFERENTIATION TRANSCRIPTION FACTOR FOR TREATMENT OF OCULAR DISORDERS

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority of Singapore Provisional Application No. 10202402129T filed on 18 July 2024, which is incorporated by reference herein in its entirety for any purpose.

SEQUENCE LISTING

[0002] This application contains a Sequence Listing that has been submitted electronically as an XML file named “88678PCT.xml”. The XML file, created on 16 July 2025, is 49,424 bytes in size. The material in the XML file is hereby incorporated by reference in its entirety.

FIELD OF INVENTION

[0003] The present disclosure relates to methods of producing ganglion progenitor cells from pluripotent stem cells and their use for treating ocular disorders. In particular, the present disclosure relates to methods of producing ganglion progenitor cells from pluripotent stem cells by transducing pluripotent stem cells with the neuronal differentiation transcription factor Neuronal Differentiation 1 NEURODI in a chemically-defined and xenogenic-free culture medium and their use for treating advanced glaucoma.

BACKGROUND

[0004] Retinal diseases characterized by irreversible loss of retinal cells are major causes of blindness worldwide. There is an unmet clinical need due to the lack of effective treatment to reverse retinal degeneration. A promising approach is to use cell replacement therapy that taps into the potential of human pluripotent stem cells. Glaucoma is one of the leading causes of global irreversible blindness and it has been estimated that 60.5 million people were affected by primary open-angle glaucoma (POAG) and primary angle-closure glaucoma (PACG) globally in 2010. The projected number of glaucoma patients worldwide will increase to 111.8 million in 2040, significantly affecting the Asians and Africans. However, there is currently no effective treatment for these advanced glaucoma patients who suffer loss of ganglion cells that could result in blindness. [0005] Conventional ganglion cell differentiation protocols used to produce ganglion cells from human pluripotent stem cells are not performed in a chemically-defined and xenogeneic free environment. Using culture environments that are not chemically defined and xenogeneic- free may lead to inconsistent and non-reproducible outcomes or end-products, rendering them unsuitable for therapeutic applications. These methods of producing ganglion cells known in the art are laborious and the protocols used generally require a long time period of about 25 days to generate the desired ganglion cells. Conventional methods also involve manipulation of multiple transcription factors such as NEUROG2, ATOH7, ISL1, POU4F2 (also known as BRN3B), and SOX4 to induce human embryonic stem cells (hESCs) to differentiate into retinal ganglion cells (RGCs). Therefore, there is a need to develop a more efficient and less labour- intensive method for producing ganglion cells from human pluripotent stem cells. There is a need to develop methods performed in a chemically-defined and xenogeneic free environment which ensures consistency and reproducibility of resulting ganglion cells, which can be used for treating ocular disorders such as advanced glaucoma.

SUMMARY

[0006] In one aspect, the present disclosure refers to a method of producing ganglion progenitor cells comprising: (a) transducing a population of pluripotent stem cells with the neuronal differentiation transcription factor Neuronal Differentiation 1 (NEURODJ) in a chemically-defined and xenogenic-free culture medium; and (b) culturing the transduced pluripotent stem cells for a period of time sufficient to allow expression of the NEURODI to thereby differentiate the transduced pluripotent stem cells into ganglion progenitor cells.

]0007] In one aspect, the present disclosure refers to a pharmaceutical composition comprising a population of retinal ganglion progenitor neuronal cells disclosed herein or a population of retinal ganglion cells disclosed herein, wherein the population of retinal ganglion progenitor neuronal cells or retinal ganglion cells are derived from the pluripotent stem cells disclosed herein.

[0008] In one aspect, the present disclosure refers to the pharmaceutical composition disclosed herein for use as a medicament.

[0009] In one aspect, the present disclosure refers to the pharmaceutical composition disclosed herein for use in treating an ocular disease. [0010] In one aspect, the present disclosure refers to a method of treating an ocular disorder, comprising administering to a subject an effective amount of the pharmaceutical composition disclosed herein.

[0011] In one aspect, the present disclosure refers to use of the pharmaceutical composition disclosed herein, in the manufacture of a medicament for treating an ocular disorder.

BRIEF DESCRIPTION OF DRAWINGS

[0012] The present disclosure will be better understood with reference to the detailed description when considered in conjunction with the non-limiting examples and the accompanying drawings, in which:

[0013] Fig. 1 (comprised of Figs. 1A, IB, and 1C) illustrates identification of NEURODI transcription factor as a candidate gene involved in early photoreceptor-ganglion lineage cell specification. Fig. 1A is a schematic diagram of laminin-based photoreceptor progenitor differentiation protocol. Hl hESCs were cultured using the photoreceptor progenitor differentiation protocol under three distinct laminin-based matrix conditions: (1) LN521 alone, (2) LN521+LN323, and (3) LN521+LN523, and differentiated cells were collected at day 32 for single-cell RNA sequencing and analysis. Fig. IB is a series of t-distributed stochastic neighbour embedding (tSNE) dimensional reduction plots showing the cells cultured on three distinct laminin-based matrix conditions at day 32 and the expression of various neuronal gene markers (e.g., VSX2, MITF, CRX, GAP43, NEFL) in these cells. Fig. 1C is a dot plot comparing expression of various neuronal gene markers (e.g., retinal gene markers) in the cells cultured on the three laminin-based matrix conditions. In the tSNE and dot plots, the labels “LN521”, “LN323”, and “LN523” refer to LN521 alone, LN521+LN323, and LN521+LN523 laminin- based matrix condition respectively. In the tSNE plots showing the gene marker expression, the grey scale represents normalised expression levels of the gene marker in individual cells calculated across all cells within specific laminin conditions/clusters and the dark coloured dots indicate higher expression of specific marker in the cells. In the dot plot, the grayscale gradient refers to its average gene expression calculated across all cells within specific laminin conditions/clusters and the size of the dot indicates the percentage of cells expressing the gene marker in each cluster.

[0014] Fig. 2 (comprised of Figs. 2A, 2B, 2C, and 2D) illustrates NEURODI as a short-listed gene candidate (GC) involved in laminin-based matrix LN521+LN523 mediated effect. The day 32 differentiated cells sent for single-cell RNA sequencing was also analysed by comparing the differentially expressed genes of the cells cultured on laminin-based matrix LN521+LN523, LN521+LN323, or LN521. Fig. 2A is a volcano plot showing the differential expression analysis of the cells cultured on LN521+LN523 laminin-based matrix, in comparison to LN521 alone and LN521+LN323 laminin-based matrix. Based on the plot, NEURODI was positively upregulated in day 32 differentiated cells cultured on laminin-based matrix LN521+LN523. The x-axis indicates the expression level fold changes (±0.5 FC indicated by vertical lines). The y-axis shows the Benjamini-Hochberg (BH) adjusted p-values of the differential expression test. The genes with positive log2 fold changes are upregulated in cells cultured on LN521+LN523 laminin-based matrix while the genes with negative log2 fold changes are upregulated in cells cultured on either LN521 alone or LN521+LN323 laminin-based matrix. Fig. 2B is a tSNE dimensional reduction plot showing cells at different cell culture timepoints (days 2, 9, 22 and 32) in the photoreceptor progenitor differentiation culture. Fig. 2C is a tSNE dimensional reduction plot showing NEURODI temporal expression at D32. The dark grey color dots indicate the cells expressing NEURODI. Fig. 2D is a violin plot showing expression level of NEURODI at various cell culture time points.

[0015] Fig. 3 (comprised of Figs. 3A, 3B, and 3C) illustrates characterization of the effects of NEURODI KO clones. Fig. 3A illustrates identification of NEURODI KO clones using PCR analysis. CRISPR guides (sgRNA-1 and sgRNA-2) were targeted to regions near the coding region of NEURODI (1071bp). Primers were designed to amplify the 1021 bp and 2109 bp sequences that are within and flanking the coding sequence of NEURODI . Three clones that had loss of NEURODI were identified (in white boxes in the gel electrophoresis blot). Fig. 3B is a scatter plot showing NEURODI KO off-target mutation identification from predicted off- target regions from CRISPOR using whole genome sequencing data. The scatter plot shows the editing efficiency of CRISPOR predicted off-target sites across SC4 (triangle shaped point), SC9 (square shaped point) and SC 19 (round shaped point) lines. The x-axis shows the chromosomes corresponding to the predicted off-target regions. The y-axis indicated the editing efficiency by comparing NEURODI KO and wild-type. The point below the horizontal dotted line had editing efficiency of less than 15%. Editing efficiency for each region was calculated by computing the percentage of reads/nucleotides edited in the KO lines with respect to the wild-type control, using GenEditEvaluator from Sentieon. Fig. 3C is a series of bar charts showing the quantitative RT-PCR analyses comparing the expression of various gene markers in Hl cells and Hl NEUROD1-KO clones after 30 days post-differentiation. Gene expression levels were normalized to the housekeeping gene (ACTB which is also known as b-actin). Normalised gene expression levels were calculated as fold change to day 2 (pluripotent cells) and are shown on a logarithmic scale. Data were expressed using the 2-AACt method and represented by mean SD. The degree of significance was assigned to the number of asterisks accordingly, whereby * p-value < 0.05, ** p-value < 0.01, *** p-value <0.001.

(0016] Fig. 4 (comprised of Figs. 4A, 4B, 4C, 4D, and 4E) illustrates characterization of the effects of NEURODI overexpression using lentiviral transduction system. Fig. 4A is a schematic diagram of NEURODI induction on hESCs. Fig. 4B shows characterisation of NEURODI overexpression in Hl hESCs fluorescence microscopy 7-days post-transduction to visualise GFP+ cells. White arrows indicate cell body; dotted white oval indicates neurites. Scale bar represents 300uM. Fig. 4C compares the relative gene expression of GFP in Hl cells that were not transduced (no virus), Hl cells that were transduced with control EF1A>EGFP construct (GFP); and EFlA>hNEURODl:T2A:EGFP (NEUROD1-GPP) 7 days posttransduction. Fig. 4D compares the relative gene expression of NEURODI in Hl cells that were not transduced (no virus), Hl cells that were transduced with control EF1A>EGFP construct (GFP); and EFlA>hNEURODl:T2A:EGFP (NEURODI -GFP) 7 days posttransduction. Gene expression levels were normalized to the housekeeping gene ACTB). Normalised gene expression levels were calculated as fold change to day 2 and are shown on a logarithmic scale. Fig. 4E shows the immunofluorescence characterisation of Hl hESCs overexpressed with EFlA>hNEURODl :T2A:EGFP viruses. Representative images are visualized via co-expression of NEURODI and EGFP fluorescence. The hESCs-derived neuronal cells expressed neuronal marker // A, TUJ1 and BRN3a (POU4F1). Cells were imaged 8 days post-transduction with EFlA>hNEURODl:T2A:EGFP viruses generated by VectorBuilder. Scale bar represents 20pm.

[0017] Fig. 5 (comprised of Figs. 5A and 5B) illustrates characterization of the effects NEURODI overexpression in induced pluripotent stem cells (iPSCs) using lentiviral transduction system. Figs. 5A and 5B show the immunofluorescence characterisation of respective Cedars-Sinai iPSCs and SCTi003-AiPSCs that were overexpressed with NEURODI using EFlA>hNEURODl :T2A:EGFP viruses. Representative images are visualized via coexpression of NEURODI and EGFP fluorescence. The iPSCs-derived neuronal cells expressed neuronal marker TUJ1 and BRN3a (POU4F1). Cells were imaged 7 days post-transduction with EFlA>hNEURODl :T2A:EGFP viruses generated by VectorBuilder. Scale bar represents 200pm. [0018] Fig. 6 illustrates single-cell transcriptional profiling of NEUROD J -over expressed Hl cells. Fig. 6 displays a series of tSNE plots showing the clusters of NEUROD 7-overexpressed Hl cells at day 9 and non-transduced cells at day 2 cultured on either LN521 alone or LN521+523 laminin-based matrix, and the expression of various neuronal gene markers (e.g., NEURODI, NEFL, NEFM, GAP43, POU4FP) in these cells. The labels “LN521” and “LN523” refer to LN521 alone and LN521+L523 laminin-based matrix condition respectively. In the tSNE plots showing the gene marker expression, the grayscale gradient represents normalised expression levels of the gene marker in individual cells calculated across all cells within specific clusters and the dark grey color dots indicate higher expression of specific marker in the cells.

[0019] Fig. 7 (comprised of Figs. 7A and 7B) illustrates single-cell transcriptional profiling of NEURODI -overexpressed Hl cells. Fig. 7A is a tSNE plot showing 10 clusters of NEUROD I -overexpressed Hl cells at day 9 and non-transduced cells at day 2 cultured on either LN521 alone orLN521+523 laminin-based matrix. Fig. 7B is a dot plot analysis showing expression of NEURODI in the 10 cell clusters identified from the tSNE plot in Fig. 7A. Based on the dot plot, clusters 6 and 9 were enriched for NEUROD 7-positive cells. The grayscale gradient refers to its average gene expression calculated across all cells within specific clusters and the size of the dot in the dot plot indicates the percentage of cells expressing NEURODI in each cluster.

[0020] Fig. 8 (comprised of Figs. 8A and 8B) illustrates single-cell transcriptional profiling of NEURODI -overexpressed Hl cells. Fig. 8A is a UMAP plot showing 10 clusters of induced NEURODI -over xpressed Hl cells at day 9 and non-transduced cells at day 2. Fig. 8B is a dot plot analysis comparing different cell type gene markers expressed in NEUROD 7-positive cells and NEUROD 7-negative cells identified from the same dataset used for generating the UMAP plot in Fig. 8A . In the dot plot, the grayscale gradient refers to its average gene expression calculated across all cells within specific clusters and the size of the dot indicates the percentage of cells expressing the gene marker in each cluster.

[0021] Fig. 9 (comprised of Figs. 9A, 9B, and 9C) illustrates single-cell transcriptional profiling of NEURODI -overexpressed Hl cells. Fig. 9A is a volcano plot showing the differential expression analysis between NEURODI -positive cells and NEURODI -negative cells. The genes with positive log2 fold changes are upregulated in NEURODI -positive cells/clusters, while the genes with negative log2 fold changes are upregulated in NEURODI- negative cells. Fig. 9B is a gene ontology analysis of differentially expressed genes between AVV/A’G/l /-positive cells and NEURODI -negative cells. The top GO biological processes overrepresented in the differentially expressed genes (FDR<0.05) between NEUR0D1- positive cells and NEURODI -negative cells are shown GeneRatio is the percentage of the differentially expressed gene counts against the total number of genes in the network. Fig. 9C is a chart showing pairwise correlations of single-cell transcriptomic expression oiNEURODl- positive and NEURO DI -negative clusters with foetal retina cells. Retina cell types were annotated by the original authors from Zuo et al., Single cell dual-omic atlas of the human developing retina, Nat Commun 15, 6792 (2024), and correlation calculated for all genes. The grayscale bar indicates the Pearson correlation coefficients.

[0022] Fig. 10 (comprised of Figs. 10A, 10B, 10C, 10D and 10E) illustrates intrinsic membrane properties of induced retinal ganglion progenitor cells with rebound-firing, nonfiring and evoked-firing activities. Fig. 10A displays exemplary traces showing the current clamp recording of a rebound-firing cell with injection of increasingly positive current from - 20 pA to 50 pA with 10 pA increment. The arrow indicated rebound action potential (AP) following membrane hyperpolarization. Figs. 10B and 10C are exemplary traces showing the respective current clamp recordings of a non-firing and evoked-firing cell. The arrow in Fig. 10C indicated evoked action potential (AP). Fig. 10D shows the AP frequency with increasing current injection for evoked-firing cells. Fig. 10E shows the resting membrane potential (RMP) of rebound-firing, non-firing and evoked-firing cells, ns indicates no significance. * p < 0.05. *** p < 0.001. Statistics was performed with unpaired Student t-test (2 -tails).

[0023] Fig. 11 (comprised of Figs. 11A, 11B, 11C, 11D, HE, HF, HG and HH) illustrates current density -voltage profile of induced retinal ganglion progenitor cells with rebound-firing, non-firing and evoked-firing activities. Fig. HA displays exemplary traces of a rebound-firing cell with injection of voltage-sensitive current from holding voltage/potential of -70 mV to different test potentials ranging from -90 mV to 50 mV with 10 mV increment. Fig. HB displays current density-voltage profile of peak inward sodium current and outward potassium current density for rebound-firing cells. Figs. HC and HD respectively shows exemplary traces and current density-voltage profile of non-firing cells. Figs. HE and HF respectively shows exemplary traces and current density -voltage profile of evoked-firing cells. In the current density-voltage profile, data presented are mean ± SEM. The current density for each cell was obtained by normalizing the peak current (pA) against the cell capacitance (pF) prior to averaging, n is the number of cells recorded. Fig. HG is a bar chart comparing maximal inward current density of rebound-firing, non-firing and evoked-firing cells. Fig. 11H is a bar chart comparing maximal outward current density of rebound-firing, non-firing and evoked- firing cells, ns indicates no significance. *** p < 0.001. Statistics was performed with unpaired Student t-test (2 -tails).

[0024] Fig. 12 (comprised of Figs. 12A and 12B) illustrates blockage of inward sodium current with 1 mM tetrodotoxin (TTX) abolished the rebound-firing activities of induced retinal ganglion progenitor cells. Fig. 12A displays exemplary traces of a rebound-firing cell showing the voltage-sensitive current evoked from holding voltage potential of -70 mV to different test potentials ranging from -90 mV to 50 mV with 10 mV increment in normal artificial cerebrospinal fluid (ACSF) (top), and during blockage of the inward sodium current with 1 mM TTX (bottom) The top arrow indicated inward sodium current during recording in normal ACSF, and the bottom arrow indicated the blockage of inward sodium current with TTX treatment. Fig. 12B displays exemplary traces of a rebound-firing cell with injection of increasingly positive current from -20 pAto 50 p A with 10 pA increment in normal ACSF (top), and during blockage of the inward sodium current with 1 mM TTX (bottom). The top arrow indicated rebound action potential during recording in normal ACSF, and the bottom arrow indicated the abolished action potential with TTX treatment.

DETAILED DESCRIPTION

[0025] The present disclosure describes a method of producing ganglion progenitor cells from pluripotent stem cells and their use for treating ocular disorders. Ganglion progenitor cells produced with the methods described herein can possess similar expression profile and/or display functional properties as those of a ganglion progenitor neuronal cell type. In addition, the ganglion progenitor cells produced with the methods described herein can be further differentiated into retinal ganglion cells for use as treatment to replace loss of ganglion cells in advanced glaucoma patients.

[0026] In a first aspect, the present disclosure refers to a method of producing ganglion progenitor cells comprising: (a) transducing a population of pluripotent stem cells with the neuronal differentiation transcription factor Neuronal Differentiation 1 (NEUR0D1 in a chemically-defined and xenogenic-free culture medium; and (b) culturing the transduced pluripotent stem cells for a period of time sufficient to allow expression of the NEURODI to thereby differentiate the transduced pluripotent stem cells into ganglion progenitor cells. [0027] In one example, the term “chemically-defined and xenogenic-free culture medium” refers to a medium that is serum-free and/or devoid of non-human contaminants. In one example, the “xenogenic-free culture medium” is a cell culture medium that is not affected by lot-to-lot variations, and contains minimal or no undefined components. In one example, the term “chemically-defined” refers to a cell culture environment where the component(s) of the cell culture matrix is quantified. In one example, where laminin is used as the cell culture matrix, the amount of pure laminin can be quantified such that the cell culture environment is “chemically-defined”. Using a “chemically-defined and xenogenic-free culture medium” reduces the likelihood of obtaining heterogeneous retinal cell types with different developmental stages. In one example, the “chemically-defined and xenogenic-free culture medium” is NutriStem hPSC XF medium (Satorius, 05-100-1 A)

[0028] As used herein, the term “NEUROD1” refers to both the gene that expresses, and the protein, Neurogenic Differentiation 1 (NEURODl/Neurodl) protein. The terms “NEURODT (with italics) and “NEURODl/Neurodl” (without italics) may be used interchangeably, and which is to be used is understood depending on the context of the present disclosure. This definition also applies to all other genes and their corresponding proteins mentioned in the present disclosure.

[0029] In one example, the method of the present disclosure comprises transducing a population of pluripotent stem cells with the neuronal differentiation transcription factor Neuronal Differentiation 1 (NEURODI) in a chemically-defined and xenogenic-free culture medium. In one example, the method of the present disclosure does not include any additional transduction step other than the transduction with NEURODI in step (a) of the first aspect. In one example, the method of the present disclosure does not include transduction of any other genes (including other neuronal differentiation transcription factors) apart from NEURODI as recited in step (a) of the first aspect In another example, the method of the present disclosure comprises transducing a population of pluripotent stem cells with the neuronal differentiation transcription factor Atonal Homolog 7 (ATOH7) in a chemically-defined and xenogenic-free culture medium. In one example, the method of the present disclosure does not include any additional transduction step other than transduction with ATOH7. In one example, the method of the present disclosure does not include transduction of any other genes (including other neuronal differentiation transcription factors) apart from ATOH7. [0030] In one example, the method of the present disclosure comprises culturing the transduced pluripotent stem cells for a period of time sufficient to allow expression of the NEURODI to thereby differentiate the transduced pluripotent stem cells into ganglion progenitor cells. In another example, the method of the present disclosure comprises culturing the transduced pluripotent stem cells for a period of time sufficient to allow expression of the ATOH7 to thereby differentiate the transduced pluripotent stem cells into ganglion progenitor cells. It was demonstrated that NEURODI is not expressed in human pluripotent stem cells based on the experimental data obtained from single cell transcriptomic analysis (Figs. 2B-2D). Data obtained from qPCR analysis also demonstrates that NEURODI is not considered to be expressed and/or present in pluripotent stem cells relative to housekeeping gene ACTB (Fig 4D) In one example, the culturing step comprises maintaining transduced pluripotent stem cells in a chemically-defined and xenogenic-free culture medium under conditions that promote cell proliferation, followed by monitoring the culture for a period sufficient to ensure NEURODI expression. In one example, expression of NEURODI can be validated by molecular assays such as, but not limited to, qPCR or single cell transcriptomic analysis. Methods of cell culture that are well known to those skilled in the art may be adapted for use in the method of the present disclosure. For example, any suitable cell culture protocol (including, but not limited to, cell culture conditions such as pH, temperature, stirring speed (if applicable), aeration (if applicable) etc.) which are known to those skilled in the art may generally be used for the cell culturing described in the present disclosure. In one example, a range of suitable substrates and/or matrices to support the growth of the cells may be employed, including, but not limited to, a matrix such as laminin and synthetic peptide-based matrix. Other matrices promoting cell attachment may also be used. Those skilled in the art will be able to readily determine the suitability of particular media and substrate/matrix combinations for use in the methods and systems of the invention using known procedures.

[0031] In one example, the method of the present disclosure further comprises identifying the ganglion progenitor cells by detecting expression of NEURODI and/or expression of one or more neuronal gene markers after culturing the transduced pluripotent stem cells for the period of time sufficient to allow expression NEURODI . In one example, the one or more neuronal gene markers are selected from the group consisting of PAX6, RGS5, ADAMTS9, ONECUT2, ONECUT1, POU4F2, GAP43, NEFM, NEFL, ATOH7, GNAT2, ARR3, RHO, RCVRN, PRDM1, NR , CRX, TUJ1, THY1, SNCG RBPMS, POU4F1 (BRN3a), and a combination thereof. In one specific example, the one or more neuronal markers for identifying the ganglion progenitor cells are PAX6 and TUJ1. In another specific example, the one or more neuronal markers for identifying the ganglion progenitor cells are PAX6, FUJI, and POU4F1 (BRN3a). In yet another specific example, the one or more neuronal markers for identifying the ganglion progenitor cells are GAP43, POU4F1 (BRN3a), and POU4F2. In one example, the method of the present disclosure comprises identifying other hESC-derived differentiated cell lines such as, but not limited to, bipolar cells, vascular cells, microglia, Muller glia, astrocytes, amacrine cells, horizontal cells, retinal pigment epithelium (RPE), retinal ganglion cells, cones, rods, photoreceptor progenitors, and retinal progenitors. In one example, the identifying is by detecting expression of one or more neuronal gene markers selected from the group consisting of TRPM1, PRKCA, GRM6, GRIK1, CAMK2B, TNFRSF10A, RGS5, CFH, CD34, TGFBR1, PILRA, C3, TRPM3, T1MP3, GLU , CM, APOD, TIAP2A, ONFCUT2, ONECUT1, LHX1, GAD1, C1QL2, TJP1, MLANA, MITF, BEST1, THY1, SNCG, RBPMS, POU4F2, POU4F1, NEFM, NEFL, GAP43, ATOH7, KIF21A, K1F2A, MCF2, MYL4, MAP4, CA2, GNA13, SLC12A6, PCBP4, ISOCI, CHRNB4, RXRG, 0TX2, THRB, RORA, PDE6H, OPN1SW, GNB3, GNGT2, GNAT2, R0M1, CRX, VSX2, ISL1, RAX, RCVRN, PDE6A, NRL, NR2E3, GNGT1, TULP1, RBP3, RP1, RORB, PRDM1, NEURODI, GUCY2D, GUCA1B, AIPL1, VIM, SOX2, SFRP2, PAX6, LHX2, and a combination thereof. In one example, expression of pluripotent markers such as, but not limited to, SOX2, POU5F1, and NANOG, is not detected in the ganglion progenitor cells and/or other hESC-derived differentiated cell lines.

[0032] In one example, the method of the present disclosure comprises seeding the pluripotent stem cells in the chemically-defined and xenogeneic-free culture medium prior to step (a) of the first aspect for a period of time sufficient to allow the pluripotent stem cells to undergo conditioning for transduction. In one example, cell seeding involves introducing cells into a culture medium at a pre-determined cell density. The cell density may affect the cell confluency (i.e., the percentage of the culture vessel surface covered by cells). Proper adherence of cells to the culture vessel surface and/or culture matrix is also important for accurate assessment of cell confluency. In one example, said period of time is one day or more from the start of said seeding. In one specific example, said period of time is two days from the start of said seeding. In one example, the term “conditioning for transduction” refers to a process of preparing cultured cells to increase their susceptibility to transduction. In one example, the process of “conditioning for transduction” involves optimizing various culture conditions to enhance the efficiency of transduction In one example, an optimal culture condition is one where the cultured cells are well-adhered to the laminin coated culture plate. In one example, an optimal condition is one where the cultured cells reach optimal confluency after cell seeding. A person skilled in the art would be well aware that the optimal cell confluency varies between different cell lines, and would be well-versed in determining the optimal cell confluency for each of the different cell lines.

[0033] In one example, the seeding described in the method of the present disclosure comprises seeding the pluripotent stem cells on a cell culture surface having a laminin matrix thereon in the culture medium. In one example, the laminin matrix comprises laminin selected from the group consisting of laminin-521 (LN521), laminin-523 (LN523), laminin-323 (LN323), or a combination thereof. In one example, the laminin matrix comprises a mixture of two laminins. In one example, the laminin matrix contains only two laminin trimers/chains/fragments, but permits other ingredients to still be present in the laminin matrix. In one example, the laminin matrix contains laminin-521 only. In one example, the laminin matrix contains laminin-523 only. In one example, the laminin matrix contains laminin-323 only. In one example, the laminin matrix contains laminin-521 and laminin-523. In one example, the laminin matrix contains laminin-521 and laminin-323. In one example, the laminin matrix contains laminin- 523 and laminin-323. In one example, the laminin matrix contains laminin-521, laminin-523, and laminin-323. In one example, the weight ratio of laminin-323/523 to laminin-521 in the laminin matrix is from about 1 :1 to about 4:1 , including from about 1 : 1 to about 2:1 (i.e. always less laminin-521 than the laminin-323/523). In particular, laminin-323 and laminin-523 are present in the external limiting membrane, which spans the rods and cones in the retina, while laminin-523 is present in Bruch's membrane found beneath the RPE. The presence of these laminins is thought to induce the differentiation of stem cells into retinal lineage-specific cells. Each laminin can be an intact protein or a protein fragment. In some examples, the laminins are intact proteins. Human recombinant laminin isoforms are found to be present within the retina, suggesting a potential recapitulation of the retinal matrix niche, and thus is considered to be more physiologically relevant compared to other types of matrices such as collagen matrix, hyaluronic acid (HA) hydrogels, and synthetic polymers.

[0034] In one example, the pluripotent stem cells used in the method of the present disclosure are mammalian pluripotent stem cells. In one example, the mammalian pluripotent stem cells include, but are not limited to, human pluripotent stem cells, rabbit pluripotent stem cells, nonhuman primate pluripotent stem cells, canine pluripotent stem cells, porcine pluripotent stem cells, bovine pluripotent stem cells, murine pluripotent stem cells (such as mouse or rat pluripotent stem cells), feline pluripotent stem cells, and ovine pluripotent stem cells. In one specific example, the mammalian pluripotent stem cells are human pluripotent stem cells. In one example, the human pluripotent stem cells are human embryonic stem cells or induced pluripotent stem cells (iPSCs). In one example, the human embryonic stem cells are Hl cells. In one example, the induced pluripotent stem cells are Cedars-Sinai iPSCs or SCTi003-AiPSCs

[0035] Methods of transduction are well known to those skilled in the art, and any suitable transduction protocol which are known to those skilled in the art may generally be used for the transduction described in the present disclosure. In one example, the transducing in step (a) of the first aspect of the present disclosure is performed using lentiviral transduction, Adeno- Associated Virus (AAV) transduction, or RNA-based gene delivery system. In one example, the transducing is performed using lentiviral transduction. A person skilled in the art would also be aware that the transduction protocols and reagents vary depending on the cell lines and/or cell culture condition used, and a person skilled in the art would be well-versed in optimizing the transduction protocol tailored to specific cell lines. In one example, the lentiviral transduction protocol involves lentiviruses generated by transfecting a population of target cells with a lentiviral vector. The population of target cells are then seeded and exposed to the lentiviruses and suitable transduction reagents well-known to a person skilled in the art. After incubation, transduced cells are selected (if applicable) and analysed for transduction efficiency and gene expression. In one example, the AAV transduction protocol involves the delivery of genetic material using AAV vectors. In one example, the RNA-based gene delivery protocol utilizes RNA molecules engineered to deliver genetic information into a population of target cells, facilitating transient or stable gene expression depending on the specific application and design of the RNA delivery system.

[0036] In one example, the detecting of the expression of NEURODI and/or the expression of the one or more neuronal markers of the method of the present disclosure is performed using a molecular biology technique, wherein the molecular biology technique is selected from the group consisting of single-cell RNA sequencing (scRNA-seq), immunofluorescence assay, reverse transcription quantitative polymerase chain reaction (RT-qPCR), Western blotting, and a combination thereof. In one example, a molecular biology technique refers to a method used to study biological molecules, such as DNA, RNA, and proteins, to understand their structure, function, and interactions. In one example, scRNA-seq enables the detection of gene expression by capturing and sequencing NEURODI and/or the one or more neuronal markers from individual cells, allowing for the quantification of RNA molecules and identification of expressed genes within a cell population. In one example, gene analysis techniques are subsequently performed on the single cell transcriptome sequencing data generated from scRNA-seq. In one example, the gene analysis techniques include but are not limited to: single cell subgroup classification, differential expression gene analysis, functional enrichment analysis, gene ontology analysis of differentially expressed genes, and pairwise correlation analysis. In one example, immunofluorescence assay detects expression of NEURODI and/or the one or more neuronal markers by using fluorescently labelled antibodies to specifically bind to the respective protein product within cells, visualizing its localization and abundance under a fluorescent microscope. In one example, RT-qPCR detects expression of NEURODI and/or the one or more neuronal markers by quantifying the amount of RNA transcribed through reverse transcription followed by real-time PCR amplification, providing a measure of gene expression levels. In one example, Western blotting detects expression of NEURODI and/or the one or more neuronal markers by separating and transferring the respective proteins from cell lysates onto a membrane, followed by specific antibody binding to the protein product of the gene, enabling visualization and quantification of the respective protein expression levels. The protocol and optimization steps required for these molecular biology techniques are well- known to a person skilled in the art. A person skilled in the art would understand that other molecular biology techniques, not limited to those listed herein, may be used accordingly for the purpose of detecting the expression of NEURODI and/or the expression of the one or more neuronal markers of the method of the present disclosure.

[0037] In one example, the detecting of the expression of NEURODI and/or the expression of the one or more neuronal markers of the method of the present disclosure is performed 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, or 24 days after step (b) of the first aspect. In one specific example, the detecting is performed 9 days after step (b) of the first aspect. A person skilled in the art would understand that the optimal number of days post-transduction to perform the detection step is dependent on the specific cell line and cell culture conditions used.

[0038] In one example, the method of the present disclosure further comprises isolating the ganglion progenitor cells after step (b) of the first aspect. In one example, the isolating is performed using Green Fluorescent Protein (GFP) isolation, antibody selection, live cell sorting by identifying surface markers, cell sorting based on cell sizes, and a combination thereof. In one example, ganglion progenitor cells expressing lentiviruses containing GFP can be isolated using GFP isolation by identifying and isolating cells containing GFP. In one example, ganglion progenitor cells can be isolated using antibody selection by targeting cell surface markers specific to these cells with fluorescently labelled antibodies, enabling their separation via fluorescence-activated cell sorting (FACS) or magnetic bead-based isolation techniques. In one example, ganglion progenitor cells can be isolated using live cell sorting by identifying surface markers specific to these cells and sorting them based on their fluorescence signal or other characteristics using flow cytometry. In one example, ganglion progenitor cells can be isolated using cell sorting based on cell sizes by gating on cells within a specific size range, typically determined by flow cytometry, to enrich for the cell population of interest. A person skilled in the art would understand that other methods of cell isolation, not limited to those listed herein, may be used accordingly for the purpose of isolating the ganglion progenitor cells of the method of the present disclosure

[0039] In one example, the ganglion progenitor cells generated from the method of the present disclosure are retinal ganglion progenitor cells (which can also be referred to as retinal ganglion progenitor neuronal cells in this disclosure).

|0040| In one example, the ganglion progenitor cells generated from the method of the present disclosure are characterized by their functionalities. In one example, the ganglion progenitor cells display similar behavioural functions/activities as a neuronal cell type. In one example, the ganglion progenitor cells exhibit neuronal activities including but are not limited to rebound-firing, non-firing and evoked-firing activities. In one example, the neuronal activities of the cells are detected using methods such as, but not limited to, whole-cell patch clamp technique which records the electrophysiological activities of the cell. A person skilled in the art would understand that other types of functional activities and/or other methods of determining functional activities, not limited to those listed herein, may be used accordingly for the purpose of characterizing the functionalities of the ganglion progenitor cells of the present disclosure.

[0041] In one example, the method of the present disclosure further comprises differentiating the isolated ganglion progenitor cells into retinal ganglion cells. In one example, the isolated ganglion progenitor cells can be differentiated into other neuronal cell types, such as those affected by Parkinson’s and/or Alzheimer’s disease such as, but not limited to, dopaminergic neuronal cells, cholinergic neuronal cells, and glutamatergic neuronal cells. In one example, the differentiating can be induced by introducing a combination of certain transcription factors or the inclusion of specific differentiation media in the cell culture. In one example, the certain transcription factors or the inclusion of specific differentiation media in the cell culture can lead to the differentiation of isolated ganglion progenitor cells into retinal ganglion cells by regulating gene expression patterns associated with the development and maturation of retinal ganglion cells. In one example, co-expression of NEURODI and certain transcription factors can induce differentiation of the isolated ganglion progenitor cells into retinal ganglion cells. Other methods known in the art can be used to induce differentiation of the isolated ganglion progenitor cells into retinal ganglion cells. In one example, addition of growth factors can induce differentiation of isolated ganglion progenitor cells into retinal ganglion cells by regulating gene expression and cellular processes essential for retinal ganglion cell development. In one example, the growth factor is brain-derived neurotrophic factor (BDNF). In one example, the growth factor is ciliary neurotrophic factor (CNTF). In one example, addition of small molecule chemical compounds that can modulate signalling pathways can be used to differentiate isolated ganglion progenitor cells into retinal ganglion cells by influencing gene expression and cellular behaviours crucial for retinal ganglion cell specification and maturation. In one example, the small molecule is N-[N-(3,5-difluorophenacetyl)-l-alanyl]-S- phenylglycine t-butyl ester (DAPT). In one example, the addition of small molecule, DAPT, can induce differentiation of isolated ganglion progenitor cells into retinal ganglion cells by inhibiting the Notch signalling pathway. In one example, induction by mechanical or physical cues, such as manipulating substrate stiffness and topography, induces the differentiation of isolated ganglion progenitor cells into retinal ganglion cells by mimicking the physiological microenvironment of the developing retina, promoting cell adhesion, morphology, and signalling pathway activation conducive for retinal ganglion cell differentiation.

[0042] In one aspect, the present disclosure refers to a phannaceutical composition comprising a population of retinal ganglion progenitor neuronal cells or a population of retinal ganglion cells as defined herein, wherein the population of retinal ganglion progenitor neuronal cells or retinal ganglion cells are derived from the pluripotent stem cells as defined herein.

[0043] In one example, the pharmaceutical composition comprising the population of retinal ganglion progenitor neuronal cells or retinal ganglion cells of the present disclosure may be administered to a subject in need thereof. Convenient modes of administration include injection (subcutaneous, intravenous, etc ). Depending on the route of administration, the pharmaceutical composition comprising population of retinal ganglion progenitor neuronal cells or retinal ganglion cells may be coated with a material to protect it from the action of enzymes, acids and other natural conditions which may inactivate its therapeutic activity. [0044] The term “pharmaceutical composition” refers to a preparation containing a pharmaceutically active ingredient which is suitable for administration to a subject The pharmaceutical composition described herein may further comprise a pharmaceutically acceptable carrier, an excipient, or a diluent. The term “pharmaceutically acceptable carrier” refers to an ingredient in a pharmaceutical composition, other than the pharmaceutically active ingredient, which is nontoxic to a subj ect. The use of such “pharmaceutically acceptable carrier” is well known in the art. A pharmaceutically acceptable carrier includes, but is not limited to, a buffer, excipient, stabilizer, or preservative. The pharmaceutical composition may be formulated for convenient and effective administration in effective amounts with a suitable pharmaceutically acceptable carrier in an acceptable dosage unit. In the case of compositions containing supplementary active ingredients, the dosages are determined by reference to the usual dose and manner of administration of the said ingredients.

[0045] In one example, the pharmaceutical composition comprising the population of retinal ganglion progenitor neuronal cells or retinal ganglion cells of the present disclosure may be administered by injection. In one example, the pharmaceutical composition comprising the population of retinal ganglion progenitor neuronal cells or retinal ganglion cells of the present disclosure may be administered by subretinal injection. In one example, the pharmaceutical composition comprising the population of retinal ganglion progenitor neuronal cells or retinal ganglion cells of the present disclosure may be administered by intravitreal injection. In the case of injectable solutions, the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyetheylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by including various anti-bacterial and/or anti-fungal agents. Suitable agents are well known to those skilled in the art and include, for example, parabens, chlorobutanol, phenol, benzyl alcohol, ascorbic acid, thimerosal, and the like. In many cases, isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, sodium chloride may be included in the pharmaceutical composition. Prolonged absorption of the injectable pharmaceutical compositions can be brought about by including in the pharmaceutical composition an agent which delays absorption, for example, aluminium monostearate and gelatin. [0046] In one aspect, the present disclosure refers to the pharmaceutical composition disclosed herein for use as a medicament.

[0047] In one aspect, the present disclosure refers to the pharmaceutical composition disclosed herein for use in treating an ocular disease.

[0048] In one aspect, the present disclosure refers to a method of treating an ocular disorder, comprising administering to a subject an effective amount of the pharmaceutical composition disclosed herein.

[0049] In one example the subject is a mammal. In one example, the term “mammal” includes, but is not limited to, humans, non-human primates, rodents (e.g., rats, mice, and guinea pigs), rabbits, cows, pigs, horses, and other mammalian species. In one specific example, the mammal is human.

[0050] In one aspect, the present disclosure refers to use of the pharmaceutical composition disclosed herein, in the manufacture of a medicament for treating an ocular disorder

[0051] In one example, the ocular disorder is an ocular disease or an optic neuropathy. In one example, the optic neuropathy is selected from the group consisting of glaucoma, optic neuritis, ischemic optic neuropathy, and Leber hereditary optic neuropathy. In one example, the ocular disorder can be secondary glaucoma which in turn can cause ocular infections. In one example, the ocular disorder can be secondary or uveitic glaucoma caused by uveitis and viral-induced anterior segment inflammation. In one example, the glaucoma is an advanced stage of glaucoma resulting in glaucoma-induced blindness. In one example, the glaucoma-induced blindness is characterized by loss of retinal ganglion cells. In one example, the term “ocular disorder” refers to any abnormal condition or disease affecting the structures of the eye, including, but not limited to, the cornea, lens, retina, optic nerve, and surrounding tissues. In one example, the term “glaucoma-induced blindness” refers to vision loss, vision impairment, or blindness caused by damage to the optic nerve associated with glaucoma. In one example, the term “loss of retinal ganglion cells” refers to degeneration or death of retinal ganglion cells located in the retina.

[0052] As used in this application, the singular form “a,” “an,” and “the” include plural references unless the context clearly dictates otherwise.

[0053] As used herein the term “treating’ or “treatment” refers to any and all uses which remedy a disease state or symptoms, prevent the establishment of disease, or otherwise prevent, hinder, retard, or reverse the progression of disease or other undesirable symptoms in any way whatsoever.

[0054] In the context of this specification, the term “an effective amount” includes within its meaning a non-toxic but sufficient amount of an agent (for example, the pharmaceutical composition comprising population of retinal ganglion progenitor neuronal cells or retinal ganglion cells of the present disclosure) to provide the desired effect. The exact amount required will vary from subject to subject depending on factors such as the species being treated, the age and general condition of the subject, the particular agent (for example, the pharmaceutical composition comprising population of retinal ganglion progenitor neuronal cells or retinal ganglion cells of the present disclosure) being administered and the mode of administration and so forth. Thus, it is not possible to specify an exact “effective amount” However, for any given case, an appropriate “effective amount” may be determined by one of skill in the art using appropriate means.

[0055] Sterile injectable solutions can be prepared by incorporating the analogue in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilisation. Generally, dispersions are prepared by incorporating the analogue into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above.

[0056] Single or multiple administrations of the pharmaceutical compositions according to the present disclosure may be carried out. One skilled in the art would be able, by appropriate means, to determine effective, non-toxic dosage levels of the pharmaceutical composition of the present disclosure and an administration pattern which would be suitable for treating the disorders, diseases and/or infections to which the pharmaceutical compositions are applicable.

[0057] Further, it will be apparent to one of ordinary skill in the art that the optimal course of treatment, such as the number of doses of the pharmaceutical composition of the present disclosure given per day for a defined number of days, can be ascertained using convention course of treatment determination tests.

[0058] As used herein, the term “increase” refers to a rise in amount, expression level or number on a positive scale. Conversely, the term “decrease” indicates a change on a negative scale. [0059] As used herein, the term “about” in the context of concentration of a substance, size of a substance, length of time, or other stated values means +/- 5% of the stated value, or +/- 4% of the stated value, or +/- 3% of the stated value, or +/- 2% of the stated value, or +/- 1% of the stated value, or +/- 0.5% of the stated value.

[0060] Throughout this disclosure, certain embodiments may be disclosed in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosed ranges. Accordingly, the description of a range should be considered to have specifically disclosed all the possible sub-ranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.

[0061] The present disclosure illustratively described herein may suitably be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein. Thus, for example, the terms "comprising", "including", "containing", etc. shall be read expansively and without limitation. Additionally, the terms and expressions employed herein have been used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the disclosure claimed. Thus, it should be understood that although the present disclosure has been specifically disclosed by preferred embodiments and optional features, modification and variation of the present disclosure embodied therein herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this present disclosure.

[0062] The disclosure has been described broadly and generically herein. Each of the narrower species and subgeneric groupings falling within the generic disclosure also form part of the present disclosure. This includes the generic description of the present disclosure with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein.

[0063] Other embodiments are within the following claims and non-limiting examples.

EXAMPLES [0064] Methods

[0065] Maintenance of human pluripotent stent cells culture

[0066] Culture plates (Costar) were coated with purified human laminin isoform overnight at 4°C at 10 pg/mL according to the manufacturer's instructions (BioLamina). Pluripotent human embryonic stem cells (hESCs) were seeded and propagated in vitro on LN-521 or LN521 + LN523 where they maintained pluripotency. The hESC line H1 (WiCell Research Institute) was cultured in a monolayer on laminin pre-coated plates and maintained in NutriStem hESC XF (Biological Industries, Israel) medium. The same conditions were also used for induced pluripotent stem cells (iPSCs) including Cedars-Sinai iPSCs and SCTi003-A iPSCs. Upon confhiency, the cells were sub-cultured by trypsinization using TrypLESelect (GIBCO Invitrogen) for 8 minutes at 37°C, 5% CO². The Hl cell line was routinely passaged at 10,000 - 20,000 cells/cm².

[0067] Laminin-based cell differentiation from human pluripotent stem cells (photoreceptor progenitor differentiation protocol)

[0068] 24-well tissue culture plates (Costar) were coated with either only LN-521; a mixture of LN-523 and LN-521 (2: 1 ratio); or a mixture of LN-323+LN-521 (2: 1 ratio). The plates were coated to obtain a final concentration of 10 pg/mL. The hESCs (Hl) were plated at a seeding density of -70,000 cells/well and maintained in NutriStem with daily change of fresh medium for two days.

[0069] The basal medium used throughout the cell differentiation process consisted of GMEM (Glasgow Minimum Essential Medium, Life Technologies) supplemented with 0.1 mM of |3- mercaptoethanol (Life Technologies), lx non-essential amino acid solution (Gibco), and 1 mM of pyruvate (Gibco).

[0070] After reaching about 70% confluency for two days, the NutriStem was replaced with neural induction media (NIM). NIM consists of 97% basal medium, 2% B27 supplement without vitamin A (Life Technologies), 1% N2 supplement (CTS, Life Technologies), 5pM SB431542 (Sigma), and 5pM of CKL7 (Sigma). NIM was changed once every two days from day 2 to day 8.

[0071] From day 9 to day 32, the cells were supplemented with photoreceptor differentiating media (PRDM), which consisted of Glasgow Minimum Essential Medium supplemented with 0.1 mM b-mercaptoethanol, IX non-essential amino acid solution, 1 mM sodium pyruvate, IX B27 supplement without vitamin A, IX N2 supplement, lOng/mL human ciliary neurotrophic factor (CNTF); Prospec-Tany Technogene, CYT-272), lOng/mL human brain-derived neurotrophic factor (BDNF; peprotech, 450-02-50), 0.5pM retinoic acid (Tocris Bioscience 0695/50) and lOpM N-[N-(3,5-difluorophenacetyl-L-alanyl)]-S-phenylglycine t-butyl ester (DAPT, Selleckchem, S2215).

[0072] Single-cell sequencing and characterization of gene temporal expression in clusters and gene expression levels

[0073] The Chromium single-cell 3’ reagent kit (lOx Genomics) was used to generate Illumina- ready sequencing libraries. The single-cell 30 libraries for day 2, day 9, day 22, and day 32 cells were generated following the manufacturer’s protocol.

[0074] For the Laminin chains single-cell (scRNA) analysis, single-cell RNA-seq datasets for photoreceptor progenitor cells derived from Hl embryonic stem cells were generated using the photoreceptor progenitor differentiation protocol under three combination of laminin chains, (1) LN521 alone, (2) LN521 + LN532, and (3) LN521 + LN323 at day 32. The RNA was isolated from each laminin chain condition using the lOx genomics kit and sequenced using the Illumina Hi-Seq3000 sequencing platform. Reads were mapped to the human genome (Ensembl version 90) and quantified using Cell Ranger lOx Genomics software. The Cell Ranger was run with the expected number of cells parameter (expect-cells) set to 10000. The output matrices (i.e. features.tsv, matrix.mtx and barcodes.tsv) were then input into R, and genes with zero counts (no expression) in all cells were discarded. Next, independent quality control tests were carried out in each lOx library. (1) cells with very low and very high library sizes (i.e., cells below and above the first and 99th percentiles of the total cell library sizes, respectively), (2) cells with low number of detected genes (i.e., cells below the fifth percentile of the total gene distribution detected in each cell), and (3) cells with more than 15% of their total gene count coming from mitochondrial genes, were removed The filtered genes across the datasets were then processed using Seurat v3 pipeline. UMI counts were normalized by regularized negative binomial regression by using the SCTransform function with default number of variable genes. In this function, the cell cycle scores previously computed for the G2M and G2 phases to regress out these effects were obtained. The RunPCA computed 50 principal components by considering just the variable genes. Next, the number of principal components to retain for t-distributed Stochastic Neighbour Embedding (tSNE) was determined using the fmdElbow function from the ChemoSpecMarkeR R package. tSNE was run using the RunTSNE function from Seurat package. The neighbourhood and clustering analyses were performed using FindNeighbors and FindClusters Seurat functions. The fmdElbow function was also run to estimate the number of principal components that could be used for Uniform Manifold Approximation and Projection (UMAP) by using the elbow method. UMAP was run using the RunUMAP (Seurat v3) function, wherein the Seurat package function FeaturePlot was used to color the expression level of marker genes. The FindMarkers function in the Seurat package was used to calculate the genes expressed in each laminin chain based on the criteria Bonferroni <0.05 and gene expressed in >25% cells. Genes that are uniquely expressed in each laminin chain were extracted using tidyverse R package. The unique gene were plotted in volcano plot for visualization using Enhanced Volcano R package. Seurat package function DotPlot was used to illustrate how gene markers were altered across different cell clusters. The grayscale gradient refers to its average gene expression calculated across all cells within specific laminin conditions/clusters, while the size of the dot represented the percentage of cells expressing the gene marker within each cluster.

[0075] A time-series single-cell RNA-seq dataset was generated for photoreceptor progenitor cells derived from Hl embryonic stem cells using the laminin protocol at days 9, 22, and 32. The analysis was conducted twice, starting from two different Hl passages. In total, eight lOx libraries (one for each time point and passage) were generated. For this, RNA was isolated using the lOx genomics kit and sequenced using the Illumina Hi-Seq3000 sequencing platform by multiplexing the eight samples in eight lanes. Reads were mapped to the human genome (Ensembl version 90) and quantified using Cell Ranger 2.1.1 lOx Genomics software. Cell Ranger with a custom-built reference transcriptome generated by filtering the Ensembl transcriptome for the gene biotypes: protein coding, lincRNA, and antisense was provided. The Cell Ranger was run with the expected number of cells parameter (expect-cells) set to 3000. It estimated 3926, 3274, 4368, 3761 , 1790, 4934, 3881, and 1938 cells for D2 P1 , D2_P2, D9_P1 , D9 P2, D22 P1, D22 P2, D32 P1, and D32 P2, respectively. In all cases, the percentage of reads that could be mapped confidently to the human genome was higher than 93%. The output matrices (i.e., genes.tsv and barcodes.tsv) were then input into R, and genes with zero counts in all cells were discarded.

[0076] Next, independent quality control tests in each lOx library was carried out. (1) cells with very low and very high library sizes (i.e., cells below and above the fifth and 99th percentiles of the total cell library sizes, respectively), (2) cells with low number of detected genes (i.e., cells below the fifth percentile of the total gene distribution detected in each cell), and (3) cells with more than 10% of their total gene count coming from mitochondrial genes, were removed. In addition, five gene quality control steps in each independent analysis were carried out: (1) only the detectable genes, which are defined as genes that could be detected with more than one transcript in at least 1% of the total cells, were taken into account; (2) genes with low average expression in the data (i.e., genes with an average expression below 0.01 were removed; this cutoff was set based on the total distribution of average gene expression across all cells and all genes); (3) genes with a high dropout rate were removed using the M3Drop 3.09 R package, which ranked all the genes between low and high false discovery rates (FDRs) and removed the bottom 25% of the genes (i.e., genes with highest dropouts); (4) outlier genes in the gene expression distributions (eg., MALAT1 gene) were removed; and (5) genes encoded by the mitochondrial genome were removed, gene counts were normalized with the scran 1.8.4 R package. Scran sizes were computed from cell pools by pre-clustering the data with the quickCluster function. The output object of this function was provided to the computeSumF actors function, following which Log2 -transformed normalized counts were computed using the normalize function in the scater 1.8.4 R package (using default parameters). Then, to account for cell cycle effects, the G2M and G1 cell cycle phase scores for each cell were computed by using the cyclone function in the scater 1.8.4 R package, to which the inventors input the set of human cell cycle genes provided in Scialdone et al (https://doi.org/10. 1016/j.ymeth.2015.06.021) Only the cell and gene sets (across the eight lOx libraries) that passed the quality control steps were considered for further analyses using the Seurat V3 pipeline. Two separate analyses were carried out, one with all the eight samples and one with only day 22 and day 32 (a total of four) samples. UMI counts were normalized by regularized negative binomial regression by using the SCTransform function with default number of variable genes. In this function, the cell cycle scores previously computed for the G2M and G2 phases to regress out these effects were provided. The RunPCA computed 50 principal components by considering just the variable genes. Next, the findElbow function from the ChemoSpecMarkeR R package was run to estimate the number of principal components that could be used for Uniform Manifold Approximation and Projection (UMAP) by using the elbow method. UMAP was run using the RunUMAP (Seurat v3) function, wherein the Seurat package function FeaturePlot was used to color the expression level of marker genes. The FindMarkers function in the Seurat package was used to calculate the differentially expressed (DE) genes between two passages based on the criteria Bonferroni <0.05, gene expressed in >10% cells, and log2 fold change >0.25 or < -0.25. The proportion of DE genes was calculated as the number of DE genes divided by the total number of expressed genes (>10% cells) at day 2, day 9, day 22, and day 32. Seurat function DotPlot was used to illustrate how marker genes were altered across different cell clusters. The size of the dot represented the percentage of cells expressing the marker gene within a cell cluster, while the color represented the average expression of the marker gene across all cells within a cell cluster. To validate the reproducibility of our photoreceptor differentiation protocol, single-cell RNA-seq datasets from Hl, H9, HS980, and HADC106 embryonic stem cells at days 9 and 32 were generated. RNAfrom these cells was isolated using the lOx genomics kit and sequenced using the Illumina Hi-Seq3000 sequencing platform. Reads were mapped using Cell Ranger and analyzed. For each cell line (Hl, H9, HS980, HADC106), the mean expression level of each gene across all the cells at day 32 was generated from individual Seurat objects. Finally, pairwise correlations of cell lines using Spearman’s ranked correlation by the heatscatter function from the LSD R package were computed.

(0077] Similar analysis using Seurat V4.3 was applied to single-cell RNA-seq dataset generated from NEURODI -overexpressed cells at day 9 and non-transduced cells at day 2.

100781 Differential Gene Expression Analysis

[0079] As discussed in the previous section, differentially expressed genes across the laminin conditions were identified using the FindMarkers function in Seurat, with significance determined by a Bonferroni -adjusted p-value threshold of <0.05 and a minimum expression threshold of 25% of cells. Genes uniquely expressed in each laminin condition were extracted and visualized using the tidyverse and EnhancedVolcano R packages, respectively.

[0080] Similarly, differential gene expression analysis was performed for NEUR0D1- overexpressed cells by comparing expression between NEUROD 7-positive and NEUROD1- negative clusters, following the same criteria for significance and cell expression. Genes uniquely expressed in these clusters were also extracted and visualized using the same methods.

[0081] Functional Analysis (Gene ontology)

[0082] Functional analysis of differentially expressed genes between NEUROD 7-positive and NEUROD -negative clusters was conducted using the enrichKEGG and gseKEGG functions from the ClusterProfiler R package. Pathways with an adjusted p-value < 0.05 were considered statistically significant. These enriched pathways were visualized using the ggplot2 and ggpubr packages (ggplot2.tidyverse.org). The cell-cell communication analysis of Neurodi clusters was performed using CellChat. [0083] Pairwise Cluster Correlations

[0084] Pairwise correlations between NEURODI -positive and NEURODI -negative clusters and foetal retinal cells were computed by generating gene specificity matrices, as described by Tosches et al., Evolution of pallium, hippocampus, and cortical cell types revealed by singlecell transcriptomics in reptiles, Science 360, 6391:881-888 (2018). Gene specificity matrices were derived by identifying common genes across clusters. For each pair of clusters from the two datasets, the specificity score for each shared gene was calculated as the ratio of gene expression within the cluster to the global gene expression. Spearman rank correlation of these specificity scores was then used to quantify the correlation between clusters across datasets. The resulting correlation heatmap was visualized using the heatmap R package (https://cran.r- project.org/web/packages/pheatmap/pheatmap.pdf).

[0085] CRISPR, CRISPOR, and PCR analysis

[0086] CRISPR knockout of NEURODI, Design and creation of guide RNAs and plasmid constructs

[0087] CRISPR-Cas9 plasmids (pX330A-Cas9-2A-GFP-lx2 and pX330S-2) were provided by Professor Shang Li (Duke-NUS). The CRISPR guide sequences were designed using an online web tool (CRISPOR.net) with the help from Ms Yeong Ming Yue (Duke-NUS). Shortlisted sgRNAs based on their on- and off-target properties, were cloned into the CRISPR/Cas9 plasmid that was tagged with GFP

[0088] Cloning of CRISPR guides into plasmids

[0089] The cloning of CRISPR guides into CRISPR-Cas9 plasmids were performed by Ms Yeong Ming Yue (Duke-NUS). The sgRNA oligonucleotides (Table 1) were phosphorylated and annealed by incubating lOOpM forward and reverse sgRNA oligonucleotides, T4 ligation buffer and T4 PNK (New England BioLabs) in a thermocycler (Bio-rad) at 37°C for 30 minutes followed by 95°C for 5 minutes and ramped to 25°C at 5°C min'¹. Pairs of the annealed oligonucleotide duplexes (MY0101 and MY0102 were annealed to form sgRNA-1 , MY0103 and MYO 104 were annealed to form sgRNA-2) were ligated into the plasmids pX330A-Cas9- 2A-GFP-lx2 and pX330S-2 respectively using the reaction mix (Table 2). The ligation reactions underwent six (6) cycles of 37°C for 5 minutes followed by 21°C for 5 minutes. After incubation, the ligation mixes were treated with Plasmidsafe exonuclease to digest residual linearised DNA before bacterial transformation. Plasmids were then extracted from the transformed bacterial cells using the miniprep or midiprep kit (Qiagen) according to manufacturer’s instructions. Sanger sequencing using the following primer 5’- GCCTTTTGCTGGCCTTTTGCTC-3’ (SEQ ID NO. 30) was used to check for correct insertion of the sgRNA in the plasmid. [0090] Table 1. Oligonucleotides sequence for sgRNA

[0091] Table 2. Reaction mix for ligation of sgRNA oligonucleotides to plasmid

[0092] Transfection and fluorescence-activated cell sorting (FACS) ofhESCs clones [0093] Hl cells were plated in 6-well culture plates and transfected using lipofectamine crisprmax cas9 transfection reagent as per manufacturer’s instructions (Life Technologies, CMAX00003) at -60% confluency. For each transfection reaction, 12ug of plasmid, 7.5uL of CRISPRMAX™ reagent were used. The GFP-expressing cells were sorted by FACS (BD FACSAria II, BD Biosciences) 48-hours post transfection and grown as single clone on 96- well culture plates.

[0094] Clone screening for NEURODI knockout

[0095] The clones were grown for about two weeks before reaching -80% confluency in the 96-well culture plates. The clones were then trypsinised and cell pellets collected and genomic DNA were extracted using DNeasy Blood & Tissue Kit (Qiagen, 69504) . Screening of the clones was carried out using RT-PCR, performed with the KAPAHiFi HotStart PCR Kit (Roche, 07958889001) and run according to the manufacturer’s instructions. NEURODI was amplified using primer pairs (Table 3). Clone samples were cycled according to the following: one cycle of 95°C for three minutes, 30 cycles of 98°C for 20 seconds, 51°C for 15 seconds, 72°C for two minutes and lastly one cycle of 72° for 2 minutes. The RT-PCR product was then run on an 1.5% agarose gel and imaged using ChemiDoc imager (Bio-rad) to identify NEURODI KO clones. Next, these NEURODI KO clones were further validated using Sanger sequencing to confirm the deletion of NEURODE

[0096] Table 3. Primer list for diagnostic PCR

[0097] Whole Genome Sequencing for NEURODI knockout clones [0098] NEURODI KO clones were assessed and validated for potential off-targets shortlisted by CRISPOR using whole genome sequencing. Whole genome sequencing data for the NEURODI KO clones were processed using the Sentieon analysis suite. To assess potential off-target effects of the knockout, gene editing efficiency was calculated using the GeneEditEvaluator tool from the Sentieon suite. Raw sequencing reads were aligned to the human genome (Ensembl version 90). The aligned BAM files, along with the amplicon regions, were provided as input to Sentieon. Editing efficiency for each region was determined by calculating the percentage of reads or nucleotides edited in the NEURODI KO clones relative to the wild-type control using GeneEditEvaluator.

[0099] Quantitative Real-time PCR (qPCR) for NEURODI knockout clones

[0100] Total RNA was extracted from cell pellets using the RNeasy Mini Kit (Qiagen, 74104). The concentration and purity of extracted RNA were determined using a NanoDrop ND-2000 spectrophotometer (NanoDrop Technologies). Total RNA (lug) was converted to cDNA using iScript (Bio-rad, 1708891). RT-qPCR was performed using the iQ SYBR Green Supermix (Bio-rad, 1708882) according to the manufacturer’s directions, with primer pairs listed in Table 4. Amplification was performed using a Bio-rad CFX96 or CFX384 (Bio-rad, USA) or QuantStudio 6 Pro (Applied Biosystems, USA) with the following cycling parameters: 40 cycles of 94°C for 15 seconds followed by 60°C for 30 seconds, and a melt curve. The cycle threshold (Ct) of the target gene was normalised to the Ct of a housekeeping gene (ACTS), and then used to calculate the relative expression as fold change to Day 2 (pluripotent cells). The relative fold-changes were calculated using the 2-AACt method and represented by mean SD. The degree of significance was assigned to the number of asterisks accordingly, whereby * p- value < 0.05, ** p-value < 0.01, *** p-value <0.001.

[0101] Table 4. Primer list for used for qPCR

[0102] Overexpression of NEURODI using lentiviral vectors

[0103] The lentiviral vectors used in this study were constructed and packaged by VectorBuilder (vectorbuilder.com). For overexpression of NEURODI, the vector pLV[Exp]EFlA>hNEURODl:T2A:EGFP (vector ID: VB230425-1788nas) was used, with vector pLV[Exp]-Puro-EFlA>EGFP (vector ID: VB900088-2243bzq) expressing eGFP only used as a control.

[0104] For transduction, Hl hESCs were plated on either LN521 alone or LN521+LN523 laminin-based matrix for 2 days before the lentivirus vectors which were diluted to 10 multiplicities of infection (MOI) in Nutristem with polybrene lOug/mL were added to the cells at 60-65% confluency. Nutristem media was changed daily, and the cells were monitored for GFP expression. Same transduction conditions were also used for overexpression of NEURODI in iPSCs.

[0105] Immunocytochemistry and imaging [0106] The transduced cells were fixed in 4% paraformaldehyde (Thermo Scientific, 28908) at room temperature for 15 minutes, following which paraformaldehyde was discarded and the cells were washed in PBS. Next, the cells were permeabilized in blocking solution containing lx PBS, 0.1% TritonX-100 (PBST), and 5% donkey serum at room temperature for 1 hour. Primary antibodies were diluted in fresh blocking solution at 1 :200 and incubated with the cells overnight at 4°C. After incubation, the primary antibodies were discarded, and the cells were washed in PBST (three 10-minutes washes) at room temperature Alexa Fluor 488- and 568- conjugated anti-rabbit/mouse/sheep secondary antibodies (Molecular Probes, A32731, A48286, A11004, A11036, A11015) were diluted in blocking solution at 1 : 1,000, and were then incubated with the cells overnight at 4°C. DAPI (Sigma, D9542), diluted at 1 :500 from stock, was added to the incubation mixture. The stained cells were then washed in PBST (three 10- minutes washes) at room temperature and ProLong Gold Antifade (Invitrogen, P36930) was added. Image acquisition was performed using LSM710 Carl Zeiss confocal microscope, and Image! was used for image analysis, including cell quantification. For cell quantification, three separate images were obtained from three different fields of view using confocal imaging. Same staining and imaging conditions were also used foriPSCs overexpressed with NEURODI .

[0107] Whole-cell patch-clamp recordings

[0108] Retinal ganglion progenitor cells derived from laminin-maintained cells overexpressing NEUROD 1 SNSTQ collected from days 7 to 9 of differentiation for patch clamp recordings. A total of 44 patch clamp readings were recorded for these cells which were selected based on morphology criteria observed with GFP expression (for e g., NEURODI overexpressed Hl cells tagged with GFP that exhibit neuronal-like morphology with small cell bodies and outgrowth of neurites).

[0109] In general, the cells were recorded with the internal solution (pipette solution) containing (in mM) 130 K-gluconate, 10 KCI, 5 EGTA, 10 HEPES, 1 MgC12, 0.5 Na3GTP, 4 Mg-ATP, 10 Na-phoshocreatine pH 7.4 (adjusted with KOH) and external solution containing (in mM): 10 Glucose, 125 NaCl, 25 NaHCO3, 1.25 NaH2PO4.2H20, 2.5 KCI, 1.8 CaC12, 1 MgC12, pH 7.4 (300-310 mOsm). The internal or external solution may also be used as the artificial cerebrospinal fluid (ACSF) solution during recordings. 1 mM tetrodotoxin (TTX) for blocking the inward sodium current was added to the solution during recording.

[0110] Whole cell recordings are performed with multiclamp 200B and 700b amplifier (Molecul r Device), low-pass filtered at 1 kHz and the series resistance was typically < 10 MQ after > 50% compensation. The P/4 protocol was used to subtract online the leak and capacitive transients.

[0111] For recording action potentials, increasing positive current was injected from -20 pAto 50 pA with 10 pA increment. For recording the inward sodium current and outward potassium current, holding voltage/potential of -70 mV to different test potentials ranging from -90 mV to 50 mV with 10 mV increment were used. The recorded current data was used to determine the current density-voltage profile of peak inward sodium current and outward potassium current density. The current density for each cell was obtained by normalizing the peak current (pA) against the cell capacitance (pF) prior to averaging. Maximal inward and outward current density were also determined from the recorded current data.

(0112] Results

[0113] Identification of NEV ROD 1 as a candidate gene for reprogramming pluripotent stem cells into ganglion progenitor cells

[0114] To investigate the transcriptional factors potentially involved in specifying progenitor cell fate during retinal development, the laminin (LN)-based photoreceptor differentiation protocol which closely mimics early stages of mammalian retinal development (Fig. 1A) was employed. This approach enables the differentiation of retinal progenitor cells from human pluripotent stem cells (hPSCs) under three distinct laminin-based matrix conditions: LN521 alone, LN521+LN323, and LN521+ LN523, until day 32 time point of differentiation. Briefly, pluripotent Hl cells were seeded at day 0 on three different laminin-based matrix conditions in the presence of NutriStem media. Differentiation began at day 2 where the media was switched to neural induction media (NIM) which was fed to the cells every 2 days for 7 days. From day 9 onwards to day 32, the cells were supplemented with photoreceptor differentiating media (PRDM).

[0115] Day 32 differentiated cells were subjected to single-cell sequencing to identify candidate genes that may be involved in reprogramming pluripotent stem cells cultured on laminin-based matrices towards photoreceptor-ganglion cell lineages specification. Differential gene analysis was then performed by comparing cells cultured on LN521+LN523 laminin- based matrix, to those cultured on LN521+LN323 and LN521 alone. Visualization of the analysis results using tSNE and dot plots revealed notable differences in gene expression profiles across the different laminin conditions. The tSNE plots showed cell populations that were mapped to day 32 cells cultured on LN521 alone (“LN521”), LN521+LN523 (“LN523”), or LN521+LN323 (“LN323”) laminin-based matrix and the expression of neuronal gene markers in these cells, whereby the cells expressing the gene markers are indicated with dark grey colour dots (Fig. IB). The dot plot compared the varied expression of neuronal gene markers (for e g., retinal cell markers) in the day 32 cells across different laminin conditions, where “LN521” refers to LN521 alone, “LN523” refers to LN521+LN523, and “LN323” refers to LN521+LN323 (Fig. 1C). The grayscale gradient in the dot plot is indicative of its average gene expression calculated across all cells within a specific cluster, while the size of the dots indicates the percentage of cells expressing the gene marker in each cluster. Both the tSNE and dot plots revealed that cells cultured on the LN521+LN523 laminin-based matrix showed significant enrichment of neuronal gene markers, and in particular, retinal ganglion progenitor markers compared to cells under the other two laminin conditions (LN521 alone and LN521+LN323 laminin-based matrix). The expression of these established markers, including GAP43, P0U4F1, and POU4F2 (Figs. IB and 1C), suggested the presence of retinal ganglion progenitor population among the day 32 differentiated cells cultured on LN521+LN523 laminin-based matrix.

[0116] Furthermore, it was found that NEURODI was positively upregulated in the day 32 cells that were differentiated to photoreceptor progenitor cells using the laminin-based matrix LN521+LN523/photoreceptor differentiation culture protocol (Fig. 2A). Single-cell cluster transcriptomes in the tSNE space allowed the separation of cells into distinct clusters, which were subsequently mapped to different time points of LN521+ LN523 laminin-based matrix cultured Hl cells (Fig. 2B). Another tSNE plot showed specific expression of NEURODI gene at day 32, where the cells expressing NEURODI are indicated with dark grey colour dots (the time point corresponds to the tSNE plot in Fig. 2B) (Fig. 2C) The violin plot showed increased expression of NEURODI at day 32 in comparison to days 2, 9 and 22 (Fig. 2D). These data showing NEURODI upregulation in the cells cultured on LN521+LN523 laminin-based matrix suggests a NEURODI may play a critical role in directing retinal ganglion or photoreceptor cell lineages during early photoreceptor-ganglion lineage cell specification.

[0117] Effects of NEURODI knockout on Hl cells

[0118] In order to understand the role of NEURODI during photoreceptor-ganglion lineage cell specification, Hl NEURODI KO clonal cell lines were generated by using CRISPR/Cas9 gene editing technology to target and delete the NEURODI coding sequence in the genome of Hl cells. Briefly, single-guide RNAs (sgRNAs) targeting regions near the coding region of NEURODI (1071bp) were designed and two specifically designed sgRNAs were shortlisted based on their on- and off-target properties and cloned into CRISPR/Cas9 plasmid that was tagged with GFP The CRISPR/Cas9 plasmid carrying the sgRNAs were transfected into Hl cells and the GFP-expressing cells were sorted by FACS (BD FACSAria II, BD Biosciences) 48-hours post transfection and grown as single clone on 96-well culture plates Single clones were expanded, and genomic DNA were extracted to test for genomic deletion of NEURODI by PCR. Several Hl NEURODI KO clones were first identified using PCR with primers designed to amplify regions within and flanking the coding sequence of NEURODI (1021bp region and 2109bp region) . Thereafter, three clones (SC4, SC9, and SC 19 clones) that exhibited the loss of NEURODI (i.e., no amplification of the 1021bp and 2109bp amplicons) were identified, as indicated in white boxes in the gel electrophoresis blot (Fig. 3A). These clones were then validated by Sanger sequencing to confirm the deletion of NEURODI . Next, whole genome sequencing was performed on the Hl NEURODI KO clones to assess and validate any off-targets from predicted off target regions shortlisted by CRISPOR. No off-target mutation was observed for all three clones and the regions where the two sgRNAs targeted were edited with >75% efficiency (Fig. 3B). This suggested high efficiency of gene editing at each on-target site (sgRNA-1 and sgRNA-2) and also corroborated the deletion of NEURODI in the Hl NERUOD1 KO clones. Additionally, quantitative RT-PCR analyses using similar neuronal gene markers that characterised the progress of the early photoreceptor progenitor developmental stages at day 2 and day 32 were conducted to compare Hl cells and Hl Neurodl- O clones after 30 days of differentiation (Fig. 3C). Gene expression levels were normalized to the housekeeping gene (ACTB, fi-actin), and normalized gene expression levels were calculated as fold change to day 2 (pluripotent cells), presented on a logarithmic scale. Data were expressed using the 2-AACt method and represented by mean SD.

[0119] Differentiation of the validated Hl NEUROD1- Q clones was performed using the photoreceptor progenitor cells differentiation protocol [method depicted in Fig. 1A and disclosed in lay et al., Photoreceptor Laminin Drives Differentiation of Human Pluripotent Stem Cells to Photoreceptor Progenitors That Partially Restore Retina Function, Molecular Therapy, 31, 3'.825-846 (2023)}, to characterise the effect of NEURODI deletion in photoreceptor development. Briefly, Hl cells and Hl NEUR0D1-K0 clones were seeded onto LN521+LN523 laminin-based matrix at -35,000 cells/cm². This timepoint was referred to as Day 0. Differentiation began at Day 2 when the cells were at -60% confluency. The protocol was split into two parts: neural induction and photoreceptor generation. For neural induction, cells were fed every two days for seven days with neural induction media (NIM), which contained Glasgow Minimum Essential Medium (GMEM; Gibco, 11710-035) supplemented with 0.1 mM b-mercaptoethanol (Life Technologies, 21985-023), IX non-essential amino acid solution (Gibco, 11140-050), 1 mM sodium pyruvate (Gibco, 11360-070), B27 supplement without vitamin A (Life Technologies, 12587001; 2%(vol/vol), N2 supplement (Life Technologies, A1370701; %(vol/vol)), 5pM SB431542 (Sigma, S4317) and 5pM CKI- 7(Sigma, C0742).

[0120] At Day 9, the cells were supplemented with photoreceptor differentiating media (PRDM), which consisted of Glasgow Minimum Essential Medium supplemented with 0.1 mM b-mercaptoethanol, IX non-essential amino acid solution, 1 mM sodium pyruvate, IX B27 supplement without vitamin A, IX N2 supplement, lOng/mL human ciliary neurotrophic factor (CNTF); Prospec-Tany Technogene, CYT-272), lOng/mL human brain-derived neurotrophic factor (BDNF; peprotech, 450-02-50), 0.5pM retinoic acid (Tocris Bioscience 0695/50) and lOpM N-[N-(3,5-difluorophenacetyl-L-alanyl)]-S-phenylglycine t-butyl ester (DAPT, Selleckchem, S2215).

[0121] At Day 32, the differentiating culture of the Hl NEURODI KO-clones maintained ablated expression of NEURODI, further corroborating the successful deletion of NEURODI and confirming that NEURODI expression was successfully knocked out (Fig. 3C). On the other hand, expression of NEURODI was found to be significantly increased (P<0.0001) in control Hl cells (without NEURODI deletion) compared to the KO-clones (SC4, SC9, and SC 19 clones). Expression of pluripotency marker OCT3/4 were downregulated as expected, after differentiation at day 32 as observed in both control and NEUR0D1-K0 clones. There were lower levels of expression for early eye-field marker, PAX6, in all the NEURODJ-KO clones, with SC4 to be significantly lower (P<0.01) compared to control. At the optic cup stage where MITE and VSX2 were supposed to be upregulated, only higher MITE expression was observed in control compared to SC19 (P<0.05). The expression of VSX2 was also significantly higher in control compared to SC4 (P<0.001) and SC19 (P<0.01) but not SC9. Gene expression of photoreceptor progenitor marker, CRX, were significantly lower in SC4 and SC 19 (P<0.01 and P<0.05 respectively) compared to control. These data taken together demonstrated that loss of NEURODI disrupts the photoreceptor-ganglion cell lineage specification which ultimately disrupt the formation of photoreceptor progenitor cells or ganglion progenitor cells.

[0122] Effects of NEURODI overexpression in lentiviral-transduced Hl cells

[0123] The Hl hESCs were seeded on two laminin-based matrix conditions: LN521 alone or LN521+LN523. The experimental construct with a EF1A promotor driving expression of human NEURODI CDS expression in frame with an eGFP tag on a lentivirus backbone was used to overexpress NEURODJ-GFP in Hl hESCs at day 2 post seeding. For control, EFl A>EGFP construct (that expressed eGFP only) was used. Constructs and lentiviruses used in the present disclosure were generated by VectorBuilder. Immunofluorescence and RT-qPCR were performed to characterise the Hl hESCs over-expressing NEURODI . Additionally, cells were collected at day 2 post-seeding (i.e., just before lentiviruses were introduced), and also day 9 post-seeding (i.e., 7 days post-transduction) for scRNA-sequencing (Fig. 4A). Fluorescence microscopy was used to visualize GFP+ cells in the Hl cells transduced either with control EFl A>EGFP or EFlA>hNE URODI :T2A:EGFP constructs at day 9 (i.e., 7-days post-transduction)(Fig. 4B). Normalized GFP and NEUROD expression levels in Hl hESCs transduced with either with control EF1A>EGFP or EFlA>hNEURODl :T2A:EGFP constructs were then measured and quantified (Figs. 4C and 4D). The results showed that Hl hESCs expressed neuronal markers PAX6 and TUJ1 (Fig. 4E).

[0124] Hl hESCs transduced with control EF1A>EGFP or EFlA>hNEURODl :T2A:EGFP constructs were maintained on laminin-based matrix in the presence of pluripotent stem cell medium, NutriStem only for 7 days post-transduction (Day 9) to eliminate any other molecules or factors that will confound the findings (Fig. 4A). By Day 9, the overexpression of the NEURODI gene in Hl cells transduced with EFlA>hNEURODl :T2A:EGFP construct resulted in a neuronal morphology, showing outgrowth of neurites, compared to cells transduced with the control EF 1 A>EGFP construct (Fig. 4B). As expected, RT-qPCR analyses demonstrated increased levels of GFP in cells transduced with both EF1 A>EGFP and EFlA>hNEURODl :T2A:EGFP constructs (Fig. 4C), while increased levels of NEURODI were only observed in cultures transduced with EFl A>hNEURODl :T2A:EGFP construct (Fig. 4D). Additionally, it was shown that Hl cells overexpressed with EFlA>hNEURODl :T2A:EGFP construct expressed the neuronal markers PAX6 and TUJ1 from immunohistochemistry analyses (Fig. 4E). Further, the neuronal gene marker BRN3a which is used to characterize ganglion progenitors has also been shown to be expressed in the H1 cells overexpressed with EFl A>hNEURODl :T2A:EGFP from the immunohistochemistry analyses, thereby suggesting the specification/development of these NEURODI overexpressing Hl cells towards the ganglion progenitor cell lineage. Together, these results demonstrated that transcription factor NEURODI alone can directly induce neuronal cell differentiation and drive the generation of retinal ganglion progenitor cells from pluripotency stage within 7 days on the laminin-based matrix isoforms in the absence of any chemical induction.

[0125] Effects of NEURODI overexpression in lentiviral-transduced SCT1003-A and

Cedars-Sinai cells [0126] The robustness of NEURODI overexpression was shown by repeating on two other iPSCs lines (SCTi003-A and Cedars-Sinai) Similar to the results of the transduced Hl cells overexpressing NEURODI, the Cedars-Sinai cells (Fig. 5A) and SCTi003-A cells (Fig. 5B) overexpressed with EFlA>hNEURODl :T2A:EGFP lentiviruses expressed the neuronal markers TUJ1 and BRN3a from immunohistochemistry analyses. These data further demonstrated that transcription factor NEURODI alone can directly induce neuronal cell differentiation and drive the generation of retinal ganglion progenitor cells from pluripotency stage on the laminin-based matrix isoforms in the absence of any chemical induction.

[0127] Single-cell transcriptional profiling data ofNEVRODl-overexpressed cells

[0128] Single-cell RNA-seq transcriptomes for retinal ganglion progenitor cells derived from Hl embryonic stem cells by over expressing NEURODI were generated. Briefly, Hl hESCs were seeded on two different laminin-based matrix conditions (LN521 alone or LN521+LN523) for 2 days until -60-65% confluency was reached and then overexpressed with NEURODI using lentiviral system for the next 7 days in the laminin-maintained conditions with the pluripotent stem cell media, NutriStem. The RNA was isolated from each laminin chain condition using the lOx genomics kit and sequenced using the Illumina Hi- Seq3000 sequencing platform. To characterize the cell populations generated upon NEURODI overexpression, scRNA-seq analysis was performed for Hl cells transduced with EFlA>hNEURODl :T2A:EGFP for 7 days, comparing them to control Hl hESCs that were not transduced with lentivirus. Single-cell cluster transcriptomes in the tSNE space allowed visualization of cell populations that were mapped to day 2 (pre-transduction) Hl cells cultured on either LN521 alone (“D2 LN521”) orLN521+LN523 laminin-based matrix (“D2 LN523”) and day 9 (post transduction) NEURODI -overexpressed Hl cells cultured on either LN521 alone (“D9_LN521”) or LN521+LN523 laminin-based matrix (“D9_LN523”), and the expression of NEURODI and various neuronal gene markers in these cells, whereby the cells expressing the gene markers are indicated with dark grey colour dots (Fig. 6). In the tSNE plots showing the expression of NEURODI and the various neuronal gene markers, NEUROD1- positive cell population showed upregulation of gene markers that characterize retinal ganglion progenitors, including GAP43, POU4F1 (BRN3a), and POU4F2, as well as axon cytoskeletal markers, NEFL and NEFM (Fig. 6).

[0129] Visualization of single-cell cluster transcriptomes in another tSNE plot displayed the separation of these cell populations into 10 distinct clusters (Fig. 7A). Notably, the dot plot comparing expression of NEURODI across various cell clusters identified in Fig. 7A showed that two clusters (Cluster 6 and Cluster 9) were enriched for NEURODI -positive cells (Fig.

7B).

[0130] Visualization of single-cell cluster transcriptomes in the UMAP space allowed the separation of cells into 10 distinct clusters that were mapped to days 2 (pre-transduction) and 9 (post-transduction) time points of Hl cells cultured on either LN521 or LN521+LN523 laminin-based matrix (Fig. 8A). The dot plot compared the expression of neuronal gene markers (including those for characterizing retinal ganglion progenitors) in both NEUR0D1- positive and NEURODI -negative cells identified from the same dataset used for generating the UMAP plot, highlighting varied expression patterns between the two groups and the upregulation of neuronal gene markers that characterized retinal ganglion progenitors in NEURODI -positive cells. (Fig. SB).

[0131] The volcano plot showed the differentially expressed genes between NEUR0D1- positive cells and N UROD I -negative cells, where genes with positive log2 fold changes are upregulated in NEURO DI -positive cells/clusters, and genes with negative fold changes are upregulated in NEUROD 1 -negative cells. In the differential expression analysis, 1098 genes were identified to be upregulated in the NEURODI -positive cells/clusters, while 1399 genes were upregulated in the NEURODI -negative clusters (Fig. 9A). Functional enrichment analysis revealed a predominant enrichment of neuronal functions among the genes upregulated in the NEURODI -positive cells/clusters, in contrast to RNA processing-related functions that were enriched in genes upregulated in the NEAJ RO DI -negative cells (Fig. 9B). Pairwise correlations of single-cell transcriptomic expression of NEURODI -positive cells and NEUROD I -negative cells with foetal retina cells were also performed, showing that NEURODI -positive cells share a similar expression profile with retinal ganglion progenitor cell types, compared to NEURODI -negative positive cells (Fig. 9C). These data further demonstrated the role of NEURODI expression in specifying ganglion progenitor cell lineage during early retinal development.

[0132] Membrane and electrophysiological properties of NEUROD -overexpressed cells

[0133] To determine whether the retinal ganglion progenitor cells derived/induced from laminin-maintained Hl cells overexpressing NEURODI are functional (i.e. whether they exhibit any functional properties of a neuronal cell type), whole-cell patch-clamp recordings are collected and analysed for these cells. A total of 44 patch clamp readings were recorded for these induced retinal ganglion progenitor cells which were selected based on morphology criteria observed with GFP expression (for e.g., NEURODI overexpressed Hl cells tagged with GFP that exhibit neuronal-like morphology with small cell bodies and outgrowth of neurites).

[0134] Interestingly, three types of activities, rebound-firing (n = 14/44), non-firing (n = 26/44) and evoked-firing (n = 4/44) were observed from the whole-cell patch clamp electrophysiological analyses, demonstrating the presence of excitable cells among the induced retinal ganglion progenitor cells. An action potential is considered "scored" when the membrane potential crosses above 0 mV. The exemplary traces showing the current clamp recordings of rebound-firing, non-firing, and evoked-firing cells detected action potential signals among the excitable cells (i.e., the rebound-firing and evoked-firing cells) (Fig. 10A, 10B, and 10C). For evoked-firing cells, the frequency of action potential is shown to increase with increasing current injection (Fig. 10D). Among the excitable cells, the resting membrane potential of cells with evoked-firing were significantly more hyperpolarized as compared to rebound-firing. (Fig. 10E). Reportedly, membrane hyperpolarization correlates with neuronal maturation suggesting a heterogenous culture of both mature and relatively immature induced retinal ganglion progenitor cells.

[0135] Notably, voltage clamp recordings showed that the maximal inward voltage-gated sodium currents were significantly smaller in non -firing cells as compared to both reboundfiring and evoked-firing cells while the outward potassium currents were not significantly different among the three groups (Figs. 11 A-11H) Blocking of the inward sodium current with tetrodotoxin (TTX) abolished the firing of action potential in the rebound-firing cells (Fig. 12A-12B), proving that initiation of action potential is dependent on the behaviour of the voltage-gated sodium channels, which is similar to the intrinsic electrical properties of a neuronal cell.

[0136] Taken together, the whole-cell patch clamp electrophysiological analyses demonstrated that reprogramming of human pluripotent stem cells on retina specific laminin isoforms using transcription factor NEURODI only can generate retinal ganglion progenitor neurons cells (RGC precursors) with functional electrophysiological activities even in the absence of chemical induction.

[0137] Discussion

[0138] The present disclosure describes a method of producing ganglion progenitor cells from pluripotent stem cells and their use for treating ocular disorders. Under specific culture conditions, the pluripotent stem cells can be reprogrammed to resemble ganglion progenitor cells, retinal ganglion progenitor cells, or retinal ganglion progenitor neuronal cells. The ganglion progenitor cells can be further differentiated into retinal ganglion cells for use as treatment to replace loss of ganglion cells in advanced glaucoma patients.

[0139] The inventors of the present disclosure have successfully developed a laminin-based photoreceptor progenitor differentiation method that generates photoreceptor progenitors after 32 days. The inventors found that transplantation of these day 32 photoreceptor progenitors was able to partially restore retina function in rodents that mimic retinitis pigmentosa patients. It was also found that the distinct laminin-based photoreceptor differentiation timepoints model the early stepwise mammalian retinal cell commitment, suggesting potential in vitro modelling system for understanding early photoreceptor development. Subsequently, the single cell transcriptomic analysis of day 32 retinal cells differentiated in different laminin isoform combinations (LN521+LN523, LN521+LN323 orLN521-only conditions) were examined and the results revealed that photoreceptor and retinal ganglion progenitor cell lineages markers were co-expressed, suggesting retinal ganglion cell specification is initiated along the specification of photoreceptor lineage. Based on the findings, the inventors identified and shortlisted gene candidates that potentially play roles in early retinal development. Transcription factor NEURODI was identified through differential transcriptomics as being highly expressed in LN521+LN523 condition at day 32 as compared to other laminin isoforms. NEURODI knockout stable hESC clonal lines were established using CR1SPR-Cas9 tools, and it was found that loss of NEURODI led to disruption of differentiation towards photoreceptor progenitor cells and as a result did not generate any photoreceptor and ganglion progenitor cells. These findings strongly support the temporal roles of NEURODI mediated by retina specific laminin isoforms. Conversely, overexpression of NEURODI through lentiviruses in hESCs resulted in neuronal cell type that resembled ganglion progenitor neuronal cells in 7 days posttransduction based on single cell transcriptomic analysis.

[0140] Previous studies have shown that retinal ganglion progenitor cells were induced within 6 days, using a combination of four transcription factors NEUR0G2, ATOH7, ISL1, and POU4F244, but such a short differentiation timeframe is likely due to the overexpression of multiple transcription factors (ATOH7, ISL1, and P0U4F2) that are well known markers of retinal ganglion cells (RGCs). In contrast, in the present disclosure, it is shown that only one transcription factor, NEURODI is sufficient to reprogram the human pluripotent stem cells which are maintained in a laminin-based system to retinal ganglion progenitor cell at an equivalent short time frame of 7 days. This strongly highlighted that the ganglion progenitor cells generated from the method in the present disclosure were solely due to NEURODI transcription factor mediated reprogramming with the support of retina specific laminin isoforms. Further, while both POU4FJ and POU4F2 are known retinal ganglion cell markers, only the increased expression of P0U4F1 but not POU4F2 was observed in the scRNAseq of the retinal ganglion progenitor cell generated in the present disclosure. This observation was also reported in a previous study where there was a dominant expression of POU4F1 instead of POU4F2 in induced RGCs. In one study that compares foetal RGCs and RGCs in organoid culture, it was found that majority of foetal RGCs expresses POU4F1, while RGCs in organoid are POU4F2 positive. Hence, the ganglion progenitor neurons of the present disclosure are likely to be more similar to foetal retinal ganglion progenitor cells as well. As described in the present disclosure, the methods of reprogramming could potentially be in the forms of lentiviruses, AAV transduction, or RNA-based gene delivery systems.

[0141] The functional assay by patch clamp electrophysiology detected action potential firing in 40% (n = 18/44) of the retinal ganglion progenitor cells generated by the present disclosure. Based on the electrophysiology analysis, while on-firing retinal ganglion progenitor cells generally display low voltage-sensitive inward sodium current and depolarized membrane potential, among excitable retinal ganglion progenitor cells, evoked-firing cells displayed significantly hyperpolarized resting membrane potential as compared to rebound firing retinal ganglion progenitor cells. Reportedly, membrane hyperpolarization correlates with neuronal maturation suggesting a heterogenous culture of both mature and relatively immature retinal ganglion progenitor cells.

[0142] Taken together, the findings in the present disclosure showed that NEURODI plays a temporal role at the early photoreceptor-ganglion cell lineage junction and that the combined effects of NEURODI with the retina specific laminin isoforms are able to generate functional firing ganglion progenitor neurons.

[0143] Therefore, the retinal ganglion progenitor cells generated by reprogramming of NEURODI can be used as treatment to replace loss of ganglion cells in advanced glaucoma patients. The hESC-derived retinal ganglion progenitor cells disclosed herein can be tested on pre-clinical animal rodent in vivo model (DBA/2J) that mimic patient glaucoma patients. One model includes the optic nerve crush rodent model. These reprogrammed ganglion progenitor cells may be applicable to optic neuropathies. [0144] The present disclosure describes for the first time:

1. A method that requires reprograming of only one transcription factor, NEURODI in human pluripotent stem cells to produce ganglion progenitor cells.

2. Amethod to produce ganglion progenitor cells from pluripotent stem cells that is performed in a chemically-defined and xenogenic-free culture environment to ensure consistent and reproducible results.

3. A simpler and less laborious method (compared to conventional methods known in the art) of producing ganglion progenitor cells from pluripotent stem cells that takes only about 9 days to complete. [0145] The method disclosed herein have the following advantages:

1. The method disclosed herein does not require long and laborious cell differentiation protocols because it requires reprogramming of only one transcription factor for a period of about 9 days to generate the desired ganglion progenitor cells.

2. The method disclosed herein involves culturing pluripotent stems cells in a pluripotent medium, which is chemically-defined and xenogeneic free to ensure homogeneity, consistency and reproducibility of the ganglion progenitor cells produced by the method disclosed herein.

SEQUENCE LISTING

Claims

Claims

1. A method of producing ganglion progenitor cells comprising:

(a) transducing a population of pluripotent stem cells with the neuronal differentiation transcription factor Neuronal Differentiation 1 (NEUR0D1) in a chemically-defined and xenogenic-free culture medium; and

(b) culturing the transduced pluripotent stem cells for a period of time sufficient to allow expression of the NEURODI to thereby differentiate the transduced pluripotent stem cells into ganglion progenitor cells.

2. The method of claim 1, further comprising identifying the ganglion progenitor cells by detecting expression of NEURODI and/or expression of one or more neuronal gene markers after culturing the transduced pluripotent stem cells for the period of time sufficient to allow expression of NEURODI, wherein optionally the one or more neuronal gene markers are selected from the group consisting of PAX6, RGS5, ADAMTS9, ONECUT2, ONECUTl, POU4F2, GAP43, NEFM, NEFF, ATOH7, GNAT2, ARR3, RHO, RCVRN, PRDM1, NRL, CRX, FUJI, THY1, SNCG, RBPMS, POU4F1, and a combination thereof, and wherein optionally the one or more neuronal markers are PAX6 and FUJI.

3. The method of claim 1 or 2, comprising seeding the pluripotent stem cells in the chemically-defined and xenogeneic-free culture medium prior to step (a) for a period of time sufficient to allow the pluripotent stem cells to undergo conditioning for transduction, wherein optionally said period of time is one day or more from the start of said seeding, and wherein optionally said period of time is two days from the start of said seeding.

4. The method of claim 3, wherein the seeding comprises seeding the pluripotent stem cells on a cell culture surface having a laminin matrix thereon in the culture medium, wherein optionally the laminin matrix comprises laminin selected from the group consisting of laminin-521, laminin-523, laminin-323, or a combination thereof.

5. The method of any one of claims 1 to 4, wherein the pluripotent stem cells are mammalian pluripotent stem cells, wherein optionally the mammalian pluripotent stem cells are human pluripotent stem cells, wherein optionally the human pluripotent stem cells are human embryonic stem cells or induced pluripotent stem cells (iPSCs), and wherein optionally the human embryonic stem cells are Hl cells.

6. The method of any one of claims 1 to 5, wherein the transducing in step (a) is performed using lentiviral transduction, Adeno-Associated Virus (AAV) transduction, or RNA-based gene delivery system, wherein optionally the transducing is performed using lentiviral transduction.

7. The method of any one of claims 2 to 6, wherein the detecting of the expression of NEURODI and/or the expression of the one or more neuronal markers is performed using a molecular biology technique, wherein the molecular biology technique is selected from the group consisting of single-cell RNA sequencing (scRNA-seq), immunofluorescence assay, reverse transcription quantitative polymerase chain reaction (RT-qPCR), Western blotting, and a combination thereof.

8. The method of any one of claims 2 to 7, wherein the detecting of the expression of NEURODI and/or the expression of the one or more neuronal markers is performed 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, or 24 days after step (b), wherein optionally the detecting is performed 9 days after step (b).

9. The method of any one of claims 1 to 8, further comprising isolating the ganglion progenitor cells after step (b).

10. The method of claim 9, wherein the isolating is performed using a technique selected from the group consisting of Green Fluorescent Protein (GFP) isolation, antibody selection, live cell sorting by identifying surface markers, cell sorting based on cell sizes, and a combination thereof.

11. The method of claim 9 or claim 10, further comprising detecting the electrophysiological activities of the ganglion progenitor cells.

12. The method of any one of claims 1-11, wherein the ganglion progenitor cells are retinal ganglion progenitor neuronal cells.

13. The method of claim 12, wherein the retinal ganglion progenitor neuronal cells exhibit rebound-firing, non-firing and/or evoked-firing activities.

14. The method of claim 9 or claim 10, further comprising differentiating the isolated ganglion progenitor cells into retinal ganglion cells.

15. A pharmaceutical composition comprising a population of retinal ganglion progenitor neuronal cells as defined in claim 12 or 13, or a population of retinal ganglion cells as defined in claim 14, wherein the population of retinal ganglion progenitor neuronal cells or retinal ganglion cells are derived from the pluripotent stem cells as defined in any one of claims 1 to 14.

16. The pharmaceutical composition of claim 15 for use as a medicament.

17. The pharmaceutical composition of claim 15 for use in treating an ocular disease.

18. A method of treating an ocular disorder, comprising administering to a subject an effective amount of the pharmaceutical composition of claim 15.

19. The method of claim 18, wherein the subject is a mammal, wherein optionally the mammal is human.

20. Use of the pharmaceutical composition of claim 15, in the manufacture of a medicament for treating an ocular disorder.

21. The pharmaceutical composition for use of claim 17, the method of claim 18 or claim 19, or the use of claim 20, wherein the ocular disorder is an ocular disease or an optic neuropathy, wherein optionally the optic neuropathy is selected from the group consisting of glaucoma, optic neuritis, ischemic optic neuropathy, and Leber hereditary optic neuropathy, wherein optionally the glaucoma is an advanced stage of glaucoma resulting in glaucoma-induced blindness, and wherein optionally the glaucoma-induced blindness is characterized by loss of retinal ganglion cells.