Cell transformation

Abstract

The present application relates to a nucleic acid molecule comprising a promoter operably linked to a nucleic acid sequence encoding myocyte enhancer factor 2C (MEF2C) or a functional variant thereof, wherein the promoter is for expression of MEF2C in macroglia. Also provided are vectors, compositions, products, methods, cells, medical uses and methods of treatment.

Description

[Technical field]

[0001] The present disclosure relates to methods and compositions for the conversion of source cells (such as Müller glia cells and astrocytes) into cone photoreceptor cells and / or retinal pigment epithelium (RPE)-like cells by introducing transcription factors, optionally one, two, three or all of MEF2C, MEF2D, RXRG and / or CRX, into the source cells, and methods for treating retinal diseases or degeneration. [Background technology]

[0002] Photoreceptors are sensory nerve cells found in all vertebrates that play a vital role in the detection and transmission of light signals, which are essential for vision. The two types of photoreceptors are cones and rods. Of the two types of photoreceptors, rods are activated in low light and cones are activated in bright light of certain wavelengths, depending on the cone expression of the photopigments rhodopsin or S / M / L opsins, respectively. Cones are mainly concentrated in the central region of the retina, the macula, and are required for central high visual acuity and color vision. Despite their importance in the retina, rod and cone cells cannot be regenerated in the event of disease, trauma or injury. Progressive loss of photoreceptors and vision due to genetic mutations (inherited retinal dystrophies), pathological damage or environmental insults leads to retinal degeneration and activation of Müller glia cells that normally support the metabolism and nutrition of retinal cell types. In developed countries, conditions such as progressive retinitis pigmentosa (RP), age-related macular degeneration (AMD) and diabetic retinopathy, all characterized by loss of photoreceptors, are the leading causes of registered blindness. AMD is one of the most common retinal degenerative disorders, mainly affecting adults over 40 years of age and accounting for 8% of all blindness worldwide. There are over 20,000 cases annually in the UK. Given the trend towards an ageing population, AMD and other retinal degenerative disorders are predicted to increase. AMD is characterized by loss of cone cells, resulting in problems with central vision and color vision. There is currently a need for treatment of such progressive retinal degenerative diseases. Currently, available treatments for wet AMD only slow the progression of the disease by inhibiting angiogenesis, but it is inevitable that people diagnosed with AMD will eventually lose their vision. With a prediction that a large proportion of the population will be affected by AMD, there is an urgent need to identify treatments that halt or reverse the loss of cone photoreceptors.

[0003] Photoreceptors convert light into electrical signals during the process of visual phototransduction. Phototransduction requires the expression of many genes that uniquely characterize photoreceptors, and understanding the complete cascade of regulatory events that control photoreceptor development remains an open question. Most studies to date have attempted to identify transcription factors expressed by retinal progenitor cells and early photoreceptors over the course of retinal development, but have not considered photoreceptor regeneration as a whole. Swaroop et al., Nature Reviews Neuroscience; pp. 563-576 (2010) discuss the transcriptional regulation of photoreceptor development and document that the balanced action of six key transcription factors (paired homeodomain transcription factor OTX2, cone-rod homeobox protein (CRX), neural retinal leucine zipper protein (NRL), photoreceptor-specific nuclear receptor (NR2E3), nuclear receptor RORβ, and thyroid hormone receptor β2 (TRβ2)) is crucial when retinal progenitor cells commit to the rod or cone lineage. WO2021 / 253078 discloses a method for generating rod photoreceptor cells from glial cells by enhancing protein expression of one or more transcription factors selected from ASCL1, NEUROD1, NRL, NR2E3, RAX, RORB, OTX2, CRX and PAX6. Specifically, Müller glial cells are reportedly reprogrammed into induced photoreceptor cells that are positive for rod photoreceptor markers.

[0004] The retinal pigment epithelium (RPE) is sandwiched between the neural retina and the choroid and serves multiple roles, including metabolic support of the retina, recycling of retinal chromophores, absorption of scattered light, and phagocytosis of shed photoreceptor outer segments. RPE cells form a cobblestone-like pigmented monolayer of polarized and highly specialized epithelial cells that are located directly adjacent to the light-sensing photoreceptors (rods and cones). Apical processes encase the photoreceptor outer segments (POS), while the highly folded RPE basement membrane attaches to Bruch's membrane, which separates the RPE from the layer of fenestrated capillaries (choriocapillaris). The morphological specialization of the RPE facilitates multiple functions of the RPE to maintain photoreceptor excitability, including transport of nutrients from the blood to the neural retina, regulation of retinal water transport, and regulation of the ionic composition of the subretinal space. Pigment-containing melanosomes of the RPE further protect retinal cells from photooxidation and absorb light scattering. Specialized phagocytosis and degradation of damaged POS tips by the RPE is essential for photoreceptor survival and for the maintenance of normal vision. Thus, loss of RPE function may ultimately lead to secondary loss of overlying photoreceptors.

[0005] Current therapeutic approaches to treat dry AMD focus on preventing or slowing disease progression and targeting known cellular pathology at early stages of the disease, with an increasing number reaching the clinical trial stage. In contrast, attempts to repopulate macular atrophy areas of the RPE have focused on cell regeneration therapy by transplantation of healthy RPE cells derived from several stem cell sources into the atrophic macular area. Zhang et al. (2014), Protein Cell, 5(1):48-58, described the direct conversion of human fibroblasts into RPE-like cells by specific factors including PAX6, OTX2, MITF, cMYC, RAX, CRX, LKF4 and NRL, of which OTX2, MITF, cMYC, RAX and CRX are said to be "crucial". Woogeng et al. (2021), Stem Cell Reports, 17, pp. 1-18, describe the derivation of human RPE-like cells from somatic tissue and report that four TFs (MITF, OTX2, LIN28, MYCL), enhanced by CRX and small molecules, were able to convert human fibroblasts into bulk cultures containing RPE-like cells. Several clinical trials are underway to treat dry AMD, however, with limited success so far.

[0006] Photoreceptor abnormality, dysfunction and / or death are the primary causes of vision impairment and blindness in most retinal disorders. RPE cell abnormality, dysfunction and / or death can also be evident in retinal diseases, such as in geographic atrophy (GA), a subsegment of late AMD. Therefore, innovative therapeutic strategies aim to replenish lost or damaged photoreceptor and / or RPE cells by cellular recruitment or cellular conversion of other cell types toward the photoreceptor and / or RPE lineage in regenerative therapies. Summary of the Invention

[0007] There remains a need in the art to provide methods for generating photoreceptor and / or RPE cells in vivo, ex vivo and in vitro as potential therapies.

[0008] Cell-based regenerative therapy using induced pluripotent stem cells or embryonic stem cells has been extensively investigated as an alternative to current treatments. However, cells such as embryonic stem cells, retinal progenitor cells, and differentiated cones are difficult to transplant into the retina due to the complex wiring between photoreceptors and interneurons. An alternative method is transdifferentiation or direct cell conversion of one cell type into another cell type without going through an intermediate pluripotent state. However, a major obstacle associated with cell transdifferentiation is the difficulty in identifying elements, such as transcription factors, required for successful cell transdifferentiation to generate targeted cells with the phenotypic and functional characteristics of the desired cells. In most cases, the necessary elements remain unknown. Current methods for identifying transcription factors required for the transdifferentiation process often rely on literature mining and trial and error methods. These approaches are inefficient and costly to actually test a set of relevant factors. Using a proprietary technology known as MOGRIFY (see, for example, WO2017106932), the inventors can efficiently identify transcription factors for converting one cell into another. The present invention relates to transcription factors predicted by the MOGRIFY version 2.5 system. In turn, regeneration of functional photoreceptors in vitro and in vivo would be of great use in the treatment of retinal diseases and retinal degeneration. Thus, there is a need in the art to develop methods that can generate functional cone photoreceptors and / or RPE cells. Advantageously, the inventors have identified methods that can transdifferentiate Müller glia cells into cone photoreceptors and / or RPE cells in vitro and in vivo using transcription factors predicted by the proprietary technology. This was unexpected because Müller glia have the ability to regenerate photoreceptors in lower vertebrates such as zebrafish, but they have limited regenerative capabilities in mammals (Nat Rev Neurosci. July 2014; 15(7); pp. 431-442). To our knowledge, Müller glia have not previously been shown to regenerate RPE cells, even in lower vertebrates.

[0009] An additional advantage of using one or more transcription factors according to the present invention is that they are shown herein to generate retinal pigment epithelium (RPE)-like cells, which can functionally support photoreceptors, thus potentially expanding the surprising therapeutic potential of one or more transcription factors according to the present invention.For example, geographic atrophy (GA), a subsegment of late AMD, is characterized by the loss of photoreceptors and retinal pigment epithelium (RPE) cells in the macula.GA affects more than 8 million people worldwide, resulting in vision loss due to macular degeneration and subsequent loss of cone cells.Currently, there is no approved treatment for GA.

[0010] A further advantage of using one or more transcription factors according to the invention is that they are shown herein to increase the levels of brain-derived neurotrophic factor (BDNF). BDNF secretion is an established function of RPE cells. BDNF has been proposed as a candidate for the treatment of neurodegenerative diseases due to its potent neuroprotective effects. This includes a proposed role in the treatment of glaucoma based on its protective effects on retinal ganglion cells (RGCs) (Kimura et al., Int. J. Mol. Sci. 2016, 17, 1584). BDNF has also been shown to protect cones against phototoxicity (Valiente-Soriano et al., Transl Vis Sci Technol. 2019 Dec. 16; 8(6):36). Without being bound by theory, the enhanced expression of BDNF thus represents a further potential mechanism by which one or more transcription factors according to the invention can provide an effective treatment for retinal diseases or degeneration.

[0011] BDNF is a known survival-promoting factor for various neural cell types, including retinal ganglion cells (RGCs). Loss of RGCs underlies glaucoma. Thus, administration of BDNF has been proposed as a therapeutic strategy for glaucoma. However, the direct usefulness of BDNF is limited by its short half-life and its inability to cross the blood-brain barrier. The data disclosed herein indicates that MEF2C, the combination of MEF2C and MEF2D with RXRG, and to a lesser extent CRX, upregulate BDNF expression in Müller glia, which may indicate their transdifferentiation into RPE-like cells. Thus, the transcription factors disclosed herein (including the nucleic acid molecules, vectors, compositions, products and cells according to the present invention) may be advantageously used to treat glaucoma.

[0012] Thus, the present invention provides a therapeutic strategy with surprising broad applicability in retinal diseases or degeneration for conditions characterized by loss or dysfunction of RPE cells and / or photoreceptors, particularly cone photoreceptors. The wide range of potential applications is made possible by the surprising ability of at least one or more transcription factors of the present invention to recruit cone-like photoreceptors and / or RPE-like cells with the associated neuroprotective effects of BDNF.

[0013] According to a first aspect, the present invention provides a nucleic acid molecule comprising a promoter operably linked to a nucleic acid sequence encoding myocyte enhancer factor 2C (MEF2C) or a functional variant thereof, wherein the promoter is for expression of MEF2C in macroglia.

[0014] According to a second aspect, the present invention provides a vector comprising a nucleic acid molecule according to the first aspect. According to a third aspect, the present invention provides a composition comprising a nucleic acid molecule according to the first aspect or a vector according to the second aspect and a pharma- ceutically acceptable carrier.

[0015] According to a fourth aspect, the present invention provides a method for producing a composition comprising the steps of: (a) a first nucleic acid molecule according to a first aspect; (b) a second nucleic acid molecule, a promoter operably linked to a nucleic acid sequence encoding one or more transcription factors selected from the group consisting of myocyte enhancer factor 2D (MEF2D), retinoid X receptor gamma (RXRG) and cone-rod homeobox (CRX), or functional variants thereof, or any combination thereof, wherein the promoter is for expression of the one or more transcription factors in macroglia; A second nucleic acid molecule; as a combined preparation for simultaneous, separate or sequential use in the treatment of retinal diseases or degeneration.

[0016] According to a fifth aspect, the present invention provides a method for converting a retinal source cell into a retinal target cell by introducing one or more transcription factors including MEF2C or functional variants thereof into the retinal source cell, thereby converting the retinal source cell into the retinal target cell.

[0017] According to a sixth aspect, the present invention provides a cell produced by the method of the fifth aspect. According to a seventh aspect, the present invention provides a nucleic acid molecule according to the first aspect, a vector according to the second aspect, a composition according to the third aspect, or a cell according to the sixth aspect for use in treating a retinal disease or retinal degeneration.

[0018] According to an eighth aspect, the present invention provides a method of treating a retinal disease or retinal degeneration in a subject, the method comprising administering a therapeutically effective amount of a nucleic acid molecule according to the first aspect, a vector according to the second aspect, a composition according to the third aspect, a product according to the fourth aspect, or a cell according to the sixth aspect to the retina of a subject in need of treatment.

[0019] Any of the features described in relation to any of the above aspects of the invention may be combined with other aspects of the invention mutatis mutandis. [Brief description of the drawings]

[0020] [Figure 1] Diagram of reprogramming Müller glia to cone photoreceptors in human retinal organoids by individual transcription factors in the MOGRIFY v.2.5 system. Successful conversion was determined by restricting expression of individual transcription factors to Müller glia using a truncated GFAP promoter (gfaABC1D) and overlaying GFAP_TFs_GFP with the cone photoreceptor specific gene ARR3. (A and B) A control lentiviral vector without transcription factor overexpression was used, where only GFP was driven by GFAP and no colocalization of GFP+ with ARR3+ cells was observed (white arrowheads exemplify where one would expect to see cell conversion and GFP / ARR3 overlay in the outer layers of the organoid if MOGRIFY transcription factors were used). When retinal organoids were transduced with individual transcription factors predicted by the MOGRIFY v.2.5 system, including MEF2C (C and D); MEF2D (E and F); and RXRG (G and H), colocalization of GFP and ARR3 was observed (white arrowheads exemplify cone photoreceptor cells marked with green GFP). Importantly, the morphology of the converted cells was consistent with photoreceptors in the outer nuclear layer, although in this case vestigial outer segments were also observed. [Diagram 2] Diagram of the design of polycistronic lentiviral vectors for MEF2C, MEF2D and RXRG. [Diagram 3]Diagram of reprogramming Müller glia to cone photoreceptors in human retinal organoids using a polycistronic lentiviral vector encoding three transcription factors (MEF2C_MEF2D_RXRG) in the MOGRIFY v.2.5 system. Expression of the three transcription factors was restricted to Müller glia using a truncated GFAP promoter and successful conversion was determined by overlaying GFAP_TF_GFP with the cone photoreceptor specific gene ARR3. (A and B) When a control lentiviral vector without transcription factor overexpression was used, no colocalization of GFAP_GFP with ARR3 was observed (white arrowheads exemplify where one would expect to see cell conversion in the outer layers of the organoid when MOGRIFY transcription factors were used). On the other hand, when we used polycistronic lentiviral vectors in human retinal organoids (C and D), transduction efficiency was qualitatively more efficient than when individual transcription factors were used, and GFP was expressed in a substantial proportion of cone photoreceptors (white arrowheads illustrate cone photoreceptor cells marked with green GFP). [Figure 4] Quantification of total cone photoreceptors (ARR3+), total GFP+ cells, and reprogrammed cones (ARR3+ / GFP+) in human retinal organoids after transduction of three transcription factors in MOGRIFY v.2.5 system. When a control vector was used, successful reprogramming of Müller glia to cones was not observed, but when a polycistronic vector encoding three transcription factors was used, we observed successful reprogramming of Müller glia to cones (ARR3+ / GFP+). [Diagram 5]Diagram of reprogramming of Müller glia to cone photoreceptors in postmortem mouse retinal explants. (A and B) When a control AAV driving mCherry expression under the GFAP promoter was used, we did not observe successful reprogramming events of Müller glia to cone photoreceptors (white arrowheads illustrate where we would expect to see cell conversion occur). Alternatively, when AAVs encoding two transcription factors predicted by the MOGRIFY v.2.5 system (MEF2D_CRX) were used (C and D), we observed colocalization of mCherry with ARR3, indicating that the transcription factors drive the conversion of Müller glia to cones. [Figure 6] Quantification of total cone photoreceptors (ARR3+), total GFP+ cells, and reprogrammed cones (ARR3+ / GFP+) in mouse retinal explants after transduction with AAVs encoding factors predicted by the MOGRIFY v.2.5 system. When a control vector was used, no successful reprogramming of Müller glia to cones was observed, however, when a bicistronic AAV vector encoding two transcription factors (MEF2c_RXRG and MEF2D_CRX) was used, we observed successful reprogramming of Müller glia to cones (ARR3+ / mCherry+). [Figure 7] 7A and 7B. Human retinal explants were tested for light responsiveness using a multi-electrode array (MEA) before and 2 weeks after transduction with either AAV_GFAP_mcherry (control, A) or AAV_GFAP_MEF2C_RXRG and AAV_GFAP_MEF2D_CRX (TF-treated, B). Two weeks after transduction with MOGRIFY's predicted transcription factors, an increased light response with blue light was observed when compared to the mCherry control that does not contain the transcription factors. [Figure 8]The number of MEA channels recording mERG was quantified. There are 60 channels on the MEA chip, however one is a reference electrode, therefore the maximum number of channels that can be recorded is 59. In A) the number of channels recording mERG responses dropped over the culture period 2 weeks after transduction when explants were treated with GFAP_mcherry control AAV, whereas in B) there is an increase in the number of channels recording mERG when explants were treated with GFAP_MEF2C_RXRG and GFAP_MEF2D_CRX AAV. [Figure 9] Indications of Müller glia transformation to cones are observed via observation of ARR3+ cells outside the outer nuclear layer following in vivo intraocular delivery of transcription factors. Immunohistochemical detection of ARR3 and mCherry in mouse retinal sections from mice treated with a bicistronic AAV vector encoding two TFs (GFAP_MEF2C-RXRG_myc and GFAP_MEF2D_CRX_mCherry) indicates that ARR3+ cells can be identified outside the photoreceptor layer (B and Bzoom). The cell bodies / nuclei of ARR3+ cells shown here reside in the INL, not the ONL, as expected. The location of the INL is consistent with that of Müller glial nuclei, thus suggesting Müller glial cells undergoing transformation. In further support of this, the cellular morphology of ARR3+ cells does not present classical cone or Müller glial morphology. Overall, this is consistent with the Müller glial reprogramming events identified in transduced retinal organoids and mouse retinal explants. Abbreviations: NFL: nerve fiber layer, INL: inner nuclear layer, ONL: outer nuclear layer. A: DAPI, B and B zoom: ARR3, C: mCherry, D: overlay of DAPI, ARR3 and mCherry. [Figure 10] FIG. 13: We observed a trend toward increased numbers of ARR3+ cells outside the outer nuclear layer (ONL) in TF-treated retinas, regardless of route of administration (subretinal vs. intravitreal). [Figure 11]Volcano plot highlighting differentially expressed genes in individual TF conditions. Shown here is a scatter plot of significance (p-value on y-axis) versus fold change in gene expression as a result of treatment with individual TFs. Controls in each case are the same as cells transduced with empty vector. Dots in the top right and top left quadrants (white with black outline) represent up- and down-regulated genes, respectively; black dots represent genes that did not show significant (p-adj<=0.05) differential (absolute log2 fold change>1) expression. Numbers of significantly up- and down-regulated genes are annotated in the right and left quadrants, respectively. [Figure 12] FIG. 11 is a heatmap guiding the identification of cell types of cells transdifferentiated in the 2D system. Each row is a gene that is significantly (p-adj<=0.05) upregulated (absolute log2 fold change>1) in one of the conditions (polycistronic, MEF2C only and CRX only) when compared to the same cells transduced with an empty vector. Shown is the normalized expression of the gene in an independent reference dataset (Cowan et al.). Along each row, darker shades represent higher expression of the gene in the corresponding cell type compared to other cell types in the Cowan et al. dataset. Similarly, the lightest shade in each row represents the least expression of the gene in the corresponding cell type compared to other cell types in the Cowan dataset. MEF2D and RXRG conditions did not have any significantly upregulated genes. [Figure 13] Heatmap of log-normalized expression of genes involved in retinal pigment epithelium (RPE) function in untransduced control, polycistronic, MEF2C only, MEF2D only, RXRG only and CRX only cells in the 2D system, where within each control and treatment condition, the three columns represent triplicates of the experiment. [Figure 14]UMAP of retinal organoid scRNAseq data from control or TF-treated retinal organoid cells. Arrowheads highlight the RPE-like population found only in the TF-treated group. [Figure 15] Boxplot diagram illustrating expression of key RPE marker genes in the same retinal organoid scRNAseq data shown in FIG. 14 across annotated cell types (x-axis) and conditions (black=control, grey=TF-treated). [Figure 16] Heatmap of normalized mean expression of genes involved in retinal pigment epithelium (RPE) function in control Müller glia and transcription factor (TF)-treated RPE-like populations. Expression data was measured by scRNAseq. [Figure 17] Box plot diagram illustrating expression of brain-derived neurotrophic factor (BDNF), a key secretory factor of retinal pigment epithelium, in cultured retinal organoid-derived Müller glia cells treated with polycistronic or monocistronic TF lentiviral vectors. Enhanced BDNF expression is seen in Müller glia cells treated with polycistronic or monocistronic MEF2C vectors compared to other individual TFs as well as non-transduced and transduced controls. CRX enhances BDNF expression, but the effect of MEF2C alone and polycistronic vector is greater than that of CRX. The enhancement of BDNF expression by polycistronic vector appears to be greater than the additive effect of the three individual transcription factors, so the enhancement may represent a synergistic effect of MEF2C, MEF2D and RXRG. The X-axis indicates cell treatment, and the Y-axis indicates BDNF expression level (transcripts per million). [Figure 18]Quantification of brain-derived neurotrophic factor (BDNF) levels secreted from retinal organoid-derived Müller glial cells treated with either a polycistronic lentiviral vector encoding three transcription factors (GFAP_MEF2C_MEF2D_RXRG_GFP) or GFP only (GFAP_GFP) in the MOGRIFY v.2.5 system. BDNF secretion was increased 4-fold in Müller glial cells treated with the polycistronic lentiviral vector compared to the GFP-only transduced control. Statistical test: unpaired t-test, p=0.0002. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0021] According to a first aspect, the present invention provides a nucleic acid molecule comprising a promoter operably linked to a nucleic acid sequence encoding myocyte enhancer factor 2C (MEF2C) or a functional variant thereof, wherein the promoter is for expression of MEF2C in macroglia.

[0022] The data disclosed herein suggests that, among the transcription factors tested, MEF2C surprisingly produces a greater number of gene expression changes after administration to Muller glial cells compared to CRX, MEF2D or RXRG.The data disclosed herein also suggests that MEF2C surprisingly has the greatest effect on RPE phagocytosis genes and BDNF expression.Therefore, MEF2C may be a single transcription factor that is useful for use in transdifferentiation into RPE-like cells and / or cone-like photoreceptors and in the treatment of related retinal diseases or retinal degeneration.

[0023] A functional variant of MEF2C can comprise an amino acid sequence at least 70% identical to SEQ ID NO:1. A functional variant of MEF2C can comprise an amino acid sequence at least 80% identical to SEQ ID NO:1. A functional variant of MEF2C can comprise an amino acid sequence at least 85% identical to SEQ ID NO:1. A functional variant of MEF2C can comprise an amino acid sequence at least 90% identical to SEQ ID NO:1. A functional variant of MEF2C can comprise an amino acid sequence at least 95% identical to SEQ ID NO:1. A functional variant of MEF2C can comprise an amino acid sequence at least 96% identical to SEQ ID NO:1. A functional variant of MEF2C can comprise an amino acid sequence at least 97% identical to SEQ ID NO:1. A functional variant of MEF2C can comprise an amino acid sequence at least 98% identical to SEQ ID NO:1. A functional variant of MEF2C can comprise an amino acid sequence at least 99% identical to SEQ ID NO:1. The nucleic acid sequence can comprise any nucleic acid sequence that encodes a functional variant of MEF2C. Thus, the nucleic acid sequence can comprise any nucleic acid sequence at least 70% identical to SEQ ID NO:1. For example, the nucleic acid sequence can include any nucleic acid sequence that is at least 90% identical to SEQ ID NO:1.

[0024] The nucleic acid sequence can include any nucleic acid sequence that encodes MEF2C. MEF2C can include an amino acid sequence according to SEQ ID NO: 1. Thus, the nucleic acid sequence can include any nucleic acid sequence that encodes SEQ ID NO: 1.

[0025] The nucleotide sequence encoding MEF2C or a functional variant thereof is (a) SEQ ID NO:3, (b) SEQ ID NO:2, or (c) SEQ ID NO:3 or a nucleotide sequence having at least 60% identity to SEQ ID NO:2 may include.

[0026] A nucleotide sequence having at least 60% identity with SEQ ID NO:3 or SEQ ID NO:2 can have at least 70%, 80%, 90% or 95% identity with SEQ ID NO:3 or SEQ ID NO:2. SEQ ID NO:3 and SEQ ID NO:2 have approximately 65% ​​sequence identity with each other and encode the same protein sequence. Thus, non-identical nucleotides may represent silent mutations (i.e., mutations that do not change the sequence of the encoded amino acids). Alternatively, non-identical nucleotides may change the amino acid sequence of the encoded amino acids, for example, by one or more conservative amino acid mutations. Non-identical nucleotides may change the amino acid sequence of the encoded amino acids by up to 20, up to 15, up to 10, up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2 or up to 1 conservative amino acid mutations.

[0027] The nucleic acid sequence can encode MEF2C. The nucleotide sequence encoding MEF2C can be (a) SEQ ID NO: 3, or (b) SEQ ID NO: 2 may include.

[0028] The nucleic acid molecule can include a promoter operably linked to a nucleic acid sequence encoding one or more transcription factors selected from the group consisting of myocyte enhancer factor 2D (MEF2D), retinoid X receptor gamma (RXRG) and cone-rod homeobox (CRX), or functional variants thereof, or any combination thereof, where the promoter is for expression of the one or more transcription factors in macroglia. The promoter operably linked to MEF2D, RXRG and / or CRX can be the same or different from the promoter operably linked to MEF2C.

[0029] The nucleic acid molecule can encode a functional variant of MEF2D, RXRG and / or CRX. A functional variant of MEF2D can comprise an amino acid sequence at least 70% identical to SEQ ID NO:4. A functional variant of MEF2D can comprise an amino acid sequence at least 80% identical to SEQ ID NO:4. A functional variant of MEF2D can comprise an amino acid sequence at least 85% identical to SEQ ID NO:4. A functional variant of MEF2D can comprise an amino acid sequence at least 90% identical to SEQ ID NO:4. A functional variant of MEF2D can comprise an amino acid sequence at least 95% identical to SEQ ID NO:4. A functional variant of MEF2D can comprise an amino acid sequence at least 96% identical to SEQ ID NO:4. A functional variant of MEF2D can comprise an amino acid sequence at least 97% identical to SEQ ID NO:4. A functional variant of MEF2D can comprise an amino acid sequence at least 98% identical to SEQ ID NO:4. A functional variant of MEF2D can comprise an amino acid sequence at least 99% identical to SEQ ID NO:4. The nucleic acid sequence can comprise any nucleic acid sequence that encodes a functional variant of MEF2D. Thus, the nucleic acid sequence can comprise any nucleic acid sequence at least 70% identical to SEQ ID NO:4. For example, the nucleic acid sequence can include any nucleic acid sequence that is at least 90% identical to SEQ ID NO:4.

[0030] The nucleic acid sequence can include any nucleic acid sequence that encodes MEF2D. MEF2D can include an amino acid sequence according to SEQ ID NO: 4. Thus, the nucleic acid sequence can include any nucleic acid sequence that encodes SEQ ID NO: 4.

[0031] The nucleotide sequence encoding MEF2D or a functional variant thereof is (a) SEQ ID NO:6, (b) SEQ ID NO:5, or (c) SEQ ID NO:6 or a nucleotide sequence having at least 60% identity to SEQ ID NO:5 may include.

[0032] A nucleotide sequence having at least 60% identity to SEQ ID NO:6 or SEQ ID NO:5 can have at least 70%, 80%, 90% or 95% identity to SEQ ID NO:6 or SEQ ID NO:5. Non-identical nucleotides may represent silent mutations (i.e., mutations that do not change the sequence of the encoded amino acids). Alternatively, non-identical nucleotides may change the amino acid sequence of the encoded amino acids, for example, by one or more conservative amino acid mutations. Non-identical nucleotides may change the amino acid sequence of the encoded amino acids by up to 20, up to 15, up to 10, up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2 or up to 1 conservative amino acid mutations.

[0033] The nucleic acid sequence can encode MEF2D. The nucleotide sequence encoding MEF2D can be (a) SEQ ID NO:6, or (b) SEQ ID NO: 5 may include.

[0034] A functional variant of RXRG can comprise an amino acid sequence at least 70% identical to SEQ ID NO:7. A functional variant of RXRG can comprise an amino acid sequence at least 80% identical to SEQ ID NO:7. A functional variant of RXRG can comprise an amino acid sequence at least 85% identical to SEQ ID NO:7. A functional variant of RXRG can comprise an amino acid sequence at least 90% identical to SEQ ID NO:7. A functional variant of RXRG can comprise an amino acid sequence at least 95% identical to SEQ ID NO:7. A functional variant of RXRG can comprise an amino acid sequence at least 96% identical to SEQ ID NO:7. A functional variant of RXRG can comprise an amino acid sequence at least 97% identical to SEQ ID NO:7. A functional variant of RXRG can comprise an amino acid sequence at least 98% identical to SEQ ID NO:7. A functional variant of RXRG can comprise an amino acid sequence at least 99% identical to SEQ ID NO:7. The nucleic acid sequence can comprise any nucleic acid sequence that encodes a functional variant of RXRG. Thus, the nucleic acid sequence can comprise any nucleic acid sequence at least 70% identical to SEQ ID NO:7. For example, the nucleic acid sequence can comprise any nucleic acid sequence at least 90% identical to SEQ ID NO:7.

[0035] The nucleic acid sequence can include any nucleic acid sequence that encodes RXRG. RXRG can include an amino acid sequence according to SEQ ID NO: 7. Thus, the nucleic acid sequence can include any nucleic acid sequence that encodes SEQ ID NO: 7.

[0036] The nucleotide sequence encoding RXRG or a functional variant thereof is (a) SEQ ID NO: 9, (b) SEQ ID NO:8, or (c) a nucleotide sequence having at least 60% identity to SEQ ID NO:9 or SEQ ID NO:8 may include.

[0037] A nucleotide sequence having at least 60% identity to SEQ ID NO:9 or SEQ ID NO:8 can have at least 70%, 80%, 90% or 95% identity to SEQ ID NO:9 or SEQ ID NO:8. Non-identical nucleotides may represent silent mutations (i.e., mutations that do not change the sequence of the encoded amino acids). Alternatively, non-identical nucleotides may change the amino acid sequence of the encoded amino acids, for example, by one or more conservative amino acid mutations. Non-identical nucleotides may change the amino acid sequence of the encoded amino acids by up to 20, up to 15, up to 10, up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2 or up to 1 conservative amino acid mutations.

[0038] The nucleic acid sequence can encode RXRG. The nucleotide sequence encoding RXRG can be (a) SEQ ID NO: 9, or (b) SEQ ID NO: 8 may include.

[0039] A functional variant of CRX can comprise an amino acid sequence at least 70% identical to SEQ ID NO: 10. A functional variant of CRX can comprise an amino acid sequence at least 80% identical to SEQ ID NO: 10. A functional variant of CRX can comprise an amino acid sequence at least 85% identical to SEQ ID NO: 10. A functional variant of CRX can comprise an amino acid sequence at least 90% identical to SEQ ID NO: 10. A functional variant of CRX can comprise an amino acid sequence at least 95% identical to SEQ ID NO: 10. A functional variant of CRX can comprise an amino acid sequence at least 96% identical to SEQ ID NO: 10. A functional variant of CRX can comprise an amino acid sequence at least 97% identical to SEQ ID NO: 10. A functional variant of CRX can comprise an amino acid sequence at least 98% identical to SEQ ID NO: 10. A functional variant of CRX can comprise an amino acid sequence at least 99% identical to SEQ ID NO: 10. The nucleic acid sequence can comprise any nucleic acid sequence that encodes a functional variant of CRX. Thus, the nucleic acid sequence can comprise any nucleic acid sequence at least 70% identical to SEQ ID NO: 10. For example, the nucleic acid sequence can include any nucleic acid sequence that is at least 90% identical to SEQ ID NO:10.

[0040] The nucleic acid sequence can include any nucleic acid sequence that encodes CRX. CRX can include an amino acid sequence according to SEQ ID NO: 10. Thus, the nucleic acid sequence can include any nucleic acid sequence that encodes SEQ ID NO: 10.

[0041] The nucleotide sequence encoding CRX or a functional variant thereof is (a) SEQ ID NO: 12, (b) SEQ ID NO: 11, or (c) a nucleotide sequence having at least 60% identity to SEQ ID NO: 12 or SEQ ID NO: 11. may include.

[0042] A nucleotide sequence having at least 60% identity to SEQ ID NO:12 or SEQ ID NO:11 can have at least 70%, 80%, 90% or 95% identity to SEQ ID NO:12 or SEQ ID NO:11. Non-identical nucleotides may represent silent mutations (i.e., mutations that do not change the sequence of the encoded amino acids). Alternatively, non-identical nucleotides may change the amino acid sequence of the encoded amino acids, for example, by one or more conservative amino acid mutations. Non-identical nucleotides may change the amino acid sequence of the encoded amino acids by up to 20, up to 15, up to 10, up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2 or up to 1 conservative amino acid mutations.

[0043] The nucleic acid sequence may encode CRX. The nucleotide sequence encoding CRX may be (a) SEQ ID NO: 12, or (b) SEQ ID NO: 11 may include.

[0044] The nucleic acid molecule can include a nucleotide sequence encoding MEF2C and a nucleotide sequence encoding RXRG. The combination of MEF2C and RXRG can mediate the conversion of Müller glia into cone-like photoreceptors, independent of MEF2D and CRX. The nucleic acid molecule can be a bicistronic molecule comprising a nucleotide sequence encoding MEF2C and a nucleotide sequence encoding RXRG. The nucleic acid molecule can encode a functional variant of MEF2C and / or a functional variant of RXRG. The nucleic acid molecule can encode a functional variant of RXRG and MEF2C. The nucleic acid molecule can encode a functional variant of MEF2C and RXRG. The nucleic acid molecule can encode a functional variant of MEF2C and RXRG. The nucleic acid molecule can encode a functional variant of MEF2C and RXRG.

[0045] The nucleic acid molecule can include a nucleotide sequence encoding MEF2C, a nucleotide sequence encoding MEF2D, and a nucleotide sequence encoding RXRG. The data disclosed herein suggests that the combination of MEF2C, MEF2D, and RXRG results in synergistic gene expression changes compared to individual transcription factors after administration to Müller glial cells. The data disclosed herein also suggests that MEF2C, MEF2D, and RXRG surprisingly upregulate various genes related to RPE function as well as BDNF expression and secretion. Thus, MEF2C, MEF2D, and RXRG can be a beneficial combination of transcription factors to be used in transdifferentiation into RPE-like cells and / or cone-like photoreceptors and in the treatment of related retinal diseases or retinal degeneration. Without being bound by theory, one or more effects of the combination of MEF2C, MEF2D, and RXRG can be observed in any combination of two of the three transcription factors. Thus, the nucleic acid molecule can include a nucleotide sequence encoding MEF2C and a nucleotide sequence encoding MEF2D. Thus, the nucleic acid molecule may comprise a nucleotide sequence encoding MEF2C and a nucleotide sequence encoding RXRG. Thus, in another embodiment, the nucleic acid molecule may comprise a nucleotide sequence encoding MEF2D and a nucleotide sequence encoding RXRG. The nucleic acid molecule may be a polycistronic molecule comprising a nucleotide sequence encoding MEF2C, a nucleotide sequence encoding MEF2D and a nucleotide sequence encoding RXRG. The nucleic acid molecule may encode one or more or a plurality of functional variants of MEF2C, MEF2D and / or RXRG. The nucleic acid molecule may encode a functional variant of MEF2D, RXRG and MEF2C. The nucleic acid molecule may encode a functional variant of MEF2C, MEF2D and RXRG. The nucleic acid molecule may encode a functional variant of MEF2C, MEF2D and RXRG. The nucleic acid molecule may encode a functional variant of MEF2C, RXRG and MEF2D. The nucleic acid molecule may encode a functional variant of MEF2D, RXRG and MEF2C.The nucleic acid molecule may encode a functional variant of MEF2C, a functional variant of MEF2D, and a functional variant of RXRG. The nucleic acid molecule may encode a functional variant of RXRG, a functional variant of MEF2C, and a functional variant of MEF2D. The nucleic acid molecule may encode a functional variant of RXRG, a functional variant of MEF2C, and a functional variant of MEF2D.

[0046] MEF2C, MEF2D and RXRG can be in any suitable arrangement within the nucleic acid molecule. The nucleic acid sequence encoding MEF2C can be 5' to the nucleic acid sequence encoding one or more additional transcription factors, such as MEF2D and / or RXRG. The nucleic acid sequence encoding RXRG can be 3' to the nucleic acid sequence encoding MEF2C. The nucleic acid sequence encoding MEF2C can be 5' to the nucleic acid sequence encoding MEF2D, and the nucleic acid sequence encoding RXRG can be 3' to the nucleic acid sequence encoding MEF2D. The nucleic acid molecule may have the following arrangement: 5'-MEF2C-MEF2D-RXRG-3'.

[0047] The nucleic acid molecule may comprise a nucleotide sequence encoding MEF2C, a nucleotide sequence encoding MEF2D, and a nucleotide sequence encoding RXRG, and optionally a nucleotide sequence encoding CRX. The nucleic acid molecule may encode one or more functional variants of MEF2C, MEF2D, and / or RXRG, and optionally CRX. The nucleic acid molecule may encode MEF2D, RXRG, a functional variant of MEF2C, and optionally CRX. The nucleic acid molecule may encode MEF2C, MEF2D, a functional variant of RXRG, and optionally CRX. The nucleic acid molecule may encode MEF2C, MEF2D, a functional variant of RXRG, and optionally CRX. The nucleic acid molecule may encode MEF2C, RXRG, a functional variant of MEF2D, and optionally CRX. The nucleic acid molecule may encode MEF2C, RXRG, a functional variant of MEF2D, and optionally CRX. The nucleic acid molecule may encode MEF2C, RXRG, a functional variant of MEF2D, and optionally CRX.

[0048] The promoter may be selected from the group consisting of glial fibrillary acidic protein (GFAP), CAR2, CD44, GLUL, PDGFRA, retinaldehyde binding protein 1 (RLBP1), S100B, SLC1A3, VIM, ProB2, GLAST, CAG and CMV. The promoter may be selected from the group consisting of GFAP, RLBP1, ProB2 and GLAST. The promoter may be a macroglia specific promoter. The macroglia may be retinal macroglia. The macroglia may be Müller glia and / or astrocytes. The macroglia may be Müller glia. The promoter may be a GFAP promoter. The promoter may be a gfaABC1D GFAP promoter. The GFAP promoter may consist of or include SEQ ID NO:13. The GFAP promoter may consist of or include SEQ ID NO:14.

[0049] According to a second aspect, the present invention provides a vector comprising a nucleic acid molecule according to the first aspect. The vector can be a viral vector. The viral vector can be selected from the group consisting of lentiviral vector, Sendai vector, herpes simplex virus (HSV) vector, adenoviral vector, adeno-associated virus (AAV) vector, episomal vector and retroviral vector. The viral vector can be selected from the group consisting of lentiviral vector, adenoviral vector and adeno-associated virus (AAV) vector. The viral vector can be an AAV vector. The viral vector can be a lentiviral vector.

[0050] The vector can be a non-viral vector.The non-viral vector can be selected from the group consisting of liposome, nanoparticle, naked DNA, plasmid and transposon.The non-viral vector can be a repRNA vector or an mRNA vector.

[0051] According to a third aspect, the present invention provides a composition comprising a nucleic acid molecule according to the first aspect or a vector according to the second aspect and a pharma- ceutically acceptable carrier. Pharmaceutically acceptable carriers suitable for delivery of compositions of the present invention and methods for their preparation will be readily apparent to those skilled in the art.

[0052] According to a fourth aspect, the present invention provides a method for producing a composition comprising the steps of: (a) a first nucleic acid molecule according to a first aspect; (b) a second nucleic acid molecule, a second nucleic acid molecule comprising a promoter operably linked to a nucleic acid sequence encoding one or more transcription factors selected from the group consisting of myocyte enhancer factor 2D (MEF2D), retinoid X receptor gamma (RXRG) and cone-rod homeobox (CRX), or functional variants thereof, or any combination thereof, wherein the promoter is for expression of the one or more transcription factors in macroglia; and as a combined preparation for simultaneous, separate or sequential use in the treatment of retinal diseases or degeneration.

[0053] The product may be one or more nucleic acid molecules encoding MEF2C or its functional variant and one or more transcription factors among the nucleic acid molecules. The one or more transcription factors may be selected from the group consisting of MEF2D, RXRG and CRX, or functional variants thereof. The one or more transcription factors may be selected from the group consisting of MEF2D and RXRG, or functional variants thereof.

[0054] The product may be one or more nucleic acid molecules encoding MEF2C and one or more transcription factors among the nucleic acid molecules. The one or more transcription factors may be selected from the group consisting of MEF2D, RXRG and CRX. The one or more transcription factors may be selected from the group consisting of MEF2D and RXRG.

[0055] At least one of the first and second nucleic acid molecules can encode MEF2D or a functional variant thereof, and at least one of the first and second nucleic acid molecules can encode RXRG or a functional variant thereof. At least one of the first and second nucleic acid molecules can encode MEF2D, and at least one of the first and second nucleic acid molecules can encode RXRG.

[0056] At least one or more of the first and second nucleic acid molecules may further encode CRX or a functional variant thereof.At least one of the first and second nucleic acid molecules may further encode CRX or a functional variant thereof.

[0057] In some cases, (a) the first nucleic acid molecule comprises a nucleic acid sequence encoding MEF2C and a nucleic acid sequence encoding RXRG; and / or (b) the second nucleic acid molecule comprises a nucleic acid sequence encoding MEF2D and a nucleic acid sequence encoding CRX.

[0058] In some cases, (a) the first nucleic acid molecule comprises a nucleic acid sequence encoding MEF2C and a nucleic acid sequence encoding RXRG; and (b) the second nucleic acid molecule comprises a nucleic acid sequence encoding MEF2D and a nucleic acid sequence encoding CRX.

[0059] The first and / or second nucleic acid molecule may be in the form of a vector or in the form of a composition. For example, the first nucleic acid molecule may be in the form of a vector of the second aspect of the invention or in the form of a composition of the third aspect of the invention. Similarly, the second nucleic acid molecule may be in the form of a vector of the second aspect of the invention or in the form of a composition of the third aspect of the invention, but in this case, the second nucleic acid molecule may not optionally code for MEF2C, but may instead code for one or more other transcription factors, which may be selected from the group consisting of MEF2D, RXRG and CRX. Any one or more of MEF2C, MEF2D, RXRG and / or CRX may be replaced by a functional variant thereof.

[0060] The product may include a third nucleic acid molecule. For example, the first, second and third nucleic acid molecules may code between them MEF2C and two or more transcription factors. The two or more transcription factors may be selected from the group consisting of MEF2D, RXRG and CRX. The two or more transcription factors may be selected from the group consisting of MEF2D and RXRG. For example, the first nucleic acid molecule may code for MEF2C, the second nucleic acid molecule may code for MEF2D, and the third nucleic acid molecule may code for RXRG. Any one or more of MEF2C, MEF2D, RXRG and / or CRX may be replaced by a functional variant thereof.

[0061] The third nucleic acid molecule may be in the form of a vector or in the form of a composition. For example, the third nucleic acid molecule may be in the form of a vector of the second aspect of the invention or in the form of a composition of the third aspect of the invention. Similarly, the third nucleic acid molecule may be in the form of a vector of the second aspect of the invention or in the form of a composition of the third aspect of the invention, but in this case, the third nucleic acid molecule may not optionally code for MEF2C, but may instead code for one or more other transcription factors, which may be selected from the group consisting of MEF2D, RXRG and CRX. Any one or more of MEF2C, MEF2D, RXRG and / or CRX may be replaced by their functional variants.

[0062] The product may include a fourth nucleic acid molecule. For example, the first, second, third and fourth nucleic acid molecules may code, between them, MEF2C and three or more transcription factors. The three or more transcription factors may be selected from the group consisting of MEF2D, RXRG and CRX. The three or more transcription factors may include MEF2D, RXRG and CRX. For example, the first nucleic acid molecule may code for MEF2C, the second nucleic acid molecule may code for MEF2D, the third nucleic acid molecule may code for RXRG and the fourth nucleic acid molecule may code for CRX. Any one or more of MEF2C, MEF2D, RXRG and / or CRX may be replaced by a functional variant thereof.

[0063] The fourth nucleic acid molecule may be in the form of a vector or in the form of a composition. For example, the fourth nucleic acid molecule may be in the form of a vector of the second aspect of the present invention, or in the form of a composition of the third aspect of the present invention. Similarly, the fourth nucleic acid molecule may be in the form of a vector of the second aspect of the present invention, or in the form of a composition of the third aspect of the present invention, but in this case, the fourth nucleic acid molecule may not code for MEF2C as needed, but may instead code for one or more other transcription factors, which may be selected from the group consisting of MEF2D, RXRG and CRX.

[0064] The first, second, third or fourth nucleic acid molecule may code for one or more of MEF2C, MEF2D, RXRG and / or CRX together in any combination. The first, second, third or fourth nucleic acid molecule may code for a functional variant of a transcription factor disclosed herein, typically instead of the corresponding transcription factor. The first, second, third or fourth nucleic acid molecule may code for one or more of MEF2C, MEF2D, RXRG and / or CRX together in any combination. The first, second, third or fourth nucleic acid molecule may code for one or more of MEF2C, MEF2D, RXRG and / or CRX together in any combination and / or may code for one or more of MEF2C, MEF2D, RXRG and / or CRX together in any combination.

[0065] The article of manufacture can, if desired, further comprise instructions for administering the formulations simultaneously, sequentially or separately to a subject in need thereof. All references herein to treatment include curative, palliative and prophylactic treatment, although it will be understood that in the context of the present invention references to preventing are more generally associated with prophylactic treatment. Treatment can also include arresting the progression of disease severity.

[0066] Mammalian, and especially human, treatment is preferred, although both human and veterinary treatments are within the scope of the invention. Treatment can include administration by intravitreal, suprachoroidal, or subretinal injection.

[0067] As used herein, the terms "combination" or "in combination with," "used in combination with," or "combination formulation" can refer to the combined administration of two or more agents simultaneously, sequentially, or separately.

[0068] As used herein, the term "simultaneous" means that two or more agents are administered concurrently, i.e., at the same time.

[0069] As used herein, the term "sequential" means that two or more agents are administered one after the other. The term "separately" as used herein means that the agents are administered independently of each other, but within a time interval that allows the agents to exhibit a combined effect, preferably a synergistic effect. Thus, administering "separately" can allow one agent to be administered, for example, within 1 minute, 5 minutes, or 10 minutes after the other agent.

[0070] According to a fifth aspect, the present invention provides a method for converting a retinal source cell into a retinal target cell by introducing one or more transcription factors including MEF2C or functional variants thereof into the retinal source cell, thereby converting the retinal source cell into the retinal target cell.

[0071] The retinal source cells may be macroglia. The retinal source cells may be Muller glial cells or astrocytes. The retinal source cells may be a population of macroglia. The retinal source cells may be a population of Muller glial cells or astrocytes.

[0072] The retinal target cells can be photoreceptor-like cells or retinal pigment epithelium (RPE)-like cells. The retinal target cells can be a population of photoreceptor-like cells or a population of retinal pigment epithelium (RPE)-like cells. The photoreceptor-like cells can be cone-like photoreceptor cells. The population of photoreceptor-like cells can be a population of cone-like photoreceptor cells.

[0073] Alternatively, the method can be described as a method of converting a source cell into a target cell by introducing one or more transcription factors, including MEF2C, or functional variants thereof into the source cell, thereby converting the source cell into the target cell. The method and / or the target cell provided by the method can be for treating retinal disease or retinal degeneration.

[0074] In some embodiments of the method, the one or more transcription factors are introduced via a nucleic acid molecule according to the first aspect, a vector according to the second aspect, a composition according to the third aspect, or a product according to the fourth aspect.

[0075] The method may be an in vitro method. The method may include culturing under suitable conditions for at least 2 days. The method may include culturing under suitable conditions for at least 4 days. The method may include culturing under suitable conditions for at least 7 days. The culturing may be a post-transduction culturing step.

[0076] According to a sixth aspect, the present invention provides a cell produced by the method of the fifth aspect. The cell can have any one or more of the characteristics of the cell produced by the method described herein. The cell can have any one or more of the characteristics of the cone-like photoreceptor described herein. The cell can have any one or more of the characteristics of the RPE-like cell described herein. The cell can be a non-natural cell. The cell can lack any one or more of the characteristics of the target cell. The cell can lack any one or more of the characteristics of the cone photoreceptor described herein. The cell can lack any one or more of the characteristics of the RPE cell described herein.

[0077] According to a seventh aspect, the present invention provides a nucleic acid molecule according to the first aspect, a vector according to the second aspect, a composition according to the third aspect, or a cell according to the sixth aspect for use in treating a retinal disease or retinal degeneration.

[0078] According to an eighth aspect, the present invention provides a method of treating a retinal disease or retinal degeneration in a subject, the method comprising administering a therapeutically effective amount of a nucleic acid molecule according to the first aspect, a vector according to the second aspect, a composition according to the third aspect, a product according to the fourth aspect, or a cell according to the sixth aspect to the retina of a subject in need of treatment.

[0079] The retinal disease or degeneration may be any retinal disease or degeneration characterized by loss of photoreceptors, particularly cone photoreceptors and / or RPE cells. The retinal disease or degeneration may be selected from the group consisting of age-related macular degeneration (AMD), retinitis pigmentosa (RP), late Best's disease, Stargut macular dystrophy, cone-rod dystrophy or glaucoma. The retinal disease or degeneration may be age-related macular degeneration (AMD). The retinal disease or degeneration may be retinitis pigmentosa (RP). The retinal disease or degeneration may be late Best's disease. The retinal disease or degeneration may be Stargut macular dystrophy. The retinal disease or degeneration may be cone-rod dystrophy. The retinal disease or degeneration may be glaucoma.

[0080] The retinal disease or retinal degeneration may be age-related macular degeneration (AMD). The AMD may be dry AMD. The dry AMD is late dry AMD. The late dry AMD may be geographic atrophy or may be characterized by geographic atrophy. The retinal disease or retinal degeneration may be geographic atrophy. The geographic atrophy may be a symptom of a retinal disease or a symptom of a retinal degeneration. The geographic atrophy may be a symptom of late dry AMD.

[0081] The present disclosure provides a method for converting source cells, such as Müller glia cells, into cone photoreceptor cells. The method for conversion includes direct cell conversion or transdifferentiation of source cells into cone photoreceptors. The inventors have confirmed that increasing the expression and / or amount of at least one or more transcription factors or variants thereof, selected from MEF2C, MEF2D and RXRG, and optionally CRX, can transdifferentiate source cells selected from Müller glia or astrocyte cells into cone photoreceptors. The method can include transdifferentiating source cells selected from Müller glia or astrocyte cells into cone-like photoreceptor cells by increasing the expression and / or amount of at least one or more transcription factors or variants thereof, including MEF2C, preferably further including MEF2D and / or RXRG, and optionally further including CRX.

[0082] The present disclosure further provides a method for converting source cells such as Müller glia cells into retinal pigment epithelium (RPE)-like cells. The method for conversion includes direct cell conversion or transdifferentiation of source cells into RPE-like cells. The inventors have confirmed that increasing the expression and / or amount of at least one or more transcription factors or their variants, including MEF2C, preferably further including MEF2D and / or RXRG, and optionally further including CRX, can transdifferentiate source cells selected from Müller glia or astrocyte cells into RPE-like cells.

[0083] As used herein, a "source cell" is a starting cell in a cell conversion process. As used by the present invention, a "source cell" may be a primary cell (non-immortalized cell) or may be derived from a cell line (immortalized cell). A source cell may also refer to a population of cells, such as a mixed population of cells, such as cells obtained from tissues, such as retinal organoids, or cells obtained from mouse or human cadaveric retinal explants. As used herein, a "source cell" is a somatic cell, which is a cell derived from one of the germ layers (ectoderm, endoderm, or mesoderm). In some embodiments, a source cell is a progenitor cell, a differentiated cell, or a transdifferentiated cell. A differentiated cell, also known as a terminally differentiated cell, does not usually have the ability to divide and generate progeny cells. A differentiated cell is a specialized cell that is designed to perform a specific role in the body. A transdifferentiated cell is a cell that is undergoing a process of transdifferentiation, i.e., the phenotype of a differentiated cell is changing toward the phenotype of another differentiated cell, but in this case the markers of a terminally differentiated cell have not yet been fully established. Preferably, the source cells according to the present invention are macroglial cells selected from the group comprising Müller glial (MG) cells and astrocyte cells.

[0084] Müller glia are a type of supporting glial cells found specifically in the retina, although they are not directly involved in the phototransduction pathway. In zebrafish (vertebrates), Müller glia can regenerate photoreceptors after retinal injury, suggesting that Müller glia are a form of adult retinal progenitor cells in lower species. Such regenerative capacity is also observed early after development in the chick retina (avian species). However, this capacity is lost in mammals, as the retina cannot regenerate after development, and therefore any injury or disease has severe consequences for vision. In 2017, Yao et al. (Nature) provided the first evidence that Müller glia can be transdifferentiated into rod photoreceptors in vivo to restore vision in blind mice. However, rods are responsible for peripheral and night vision, whereas humans are much more dependent on cone photoreceptors for central vision, and therefore there is a greater need to identify means to transdifferentiate Müller glia into cone photoreceptors for regenerative approaches. In this regard, no one has used Müller glia cells to recreate cone photoreceptor cells. Such a method has a tremendous advantage over the use of other cells, such as artificial stem cells or embryonic stem cells, because Müller glia cells can be transformed in vivo, thus eliminating any possibility of rejection problems, and because they are already in situ, they have the interconnection pathways between other cells of the retinal tissue, which are crucial for the functional operation of the cells.

[0085] As used herein, "Müller glial cells" refers to any cell that has the characteristics of a Müller glial cell. This may be based on the presence of at least one marker and / or at least one morphological feature of Müller glial cells. The at least one marker of Müller glial cells is selected from glial fibrillary acidic protein (GFAP), Mu crystallin homolog (CRYM), or glutamine synthetase (GLUL), or a combination thereof. In the intact retina, the nuclei of Müller glial cells are anatomically located in the INL (inner nuclear layer), while the cells are spread throughout the neural retina. Müller glial cells may be healthy cells or may be activated through diseases such as inherited retinal degeneration or age-related macular degeneration. In retinal degeneration, the loss of photoreceptors leads to reactive gliosis, upregulation of GFAP, and proliferation of Müller glial cells. The Müller glial cells or populations of Müller glial cells of the present invention are typically mammalian cells, such as human cells, primate cells, rodent cells (e.g. mouse or rat) and bovine cells, preferably human cells. "Müller glial cells" as used in accordance with the present invention encompass Müller glial cells or populations of Müller glial cells.

[0086] Astrocytes are a type of glial cell that, unlike Müller glia, do not originate from retinal progenitor cells, but are similarly derived from the neuroectodermal lineage. Müller glia and astrocytes form a population of cells known to be macroglia that are distinct from microglia in origin and function. Retinal astrocytes originate from the brain and optic nerve and invade the retina during development, and therefore are not present in human retinal organoids. Astrocytes constitutively express the intermediate filament glial fibrillary acidic protein (GFAP), but this GFAP is only expressed in Müller glial cells upon activation through injury or disease. It is therefore believed that the method according to the present invention will similarly convert astrocytes in the human retina to express GFAP, and also overexpress exogenous transcription factors.

[0087] Photoreceptors are located in the neural retina, which lines the back of the eye. Photoreceptors respond to light and play a vital role in vision. There are two types of photoreceptors: cone cells and rod cells. Rod photoreceptors are highly sensitive in dim light and function to mediate night vision; rod photoreceptors use rhodopsin as the light-sensitive photopigment. Cone photoreceptors exist as several subtypes, each of which contains a specific opsin that is maximally sensitive to a different wavelength of light; thus, cones mediate color vision. Depending on the expression of opsins, there are three subtypes of cone cells: (short, mid, long wavelength; blue, green, and red). Cone cells are fewer in number and cone-shaped compared to rod cells. Both cones and rods are located in the ONL (outer nuclear layer) of the retina. Of the two types of photoreceptors, cone cells are crucial for central, high-vision vision in humans.

[0088] A "cone photoreceptor cell" or "cone-like photoreceptor cell" as used in this disclosure is any cell that has the characteristics of a cone photoreceptor cell. This may be based on the presence of at least one marker and / or at least one morphological feature of a cone photoreceptor cell. The morphology of cones is well described in the literature (Daniele et al., IOVS 2005); cone photoreceptor cells exhibit tightly packed disks rich in photopigments (opsins) that make up their outer segments. The inner segments contain numerous mitochondria, and have synaptic terminals that interact with interneurons to transmit phototransduction signals. At least one specific marker of a cone photoreceptor cell is selected from one of cone arrestin (ARR3), GNAT2, and S / M / L opsins, and combinations thereof. At least one morphological feature of a cone photoreceptor cell is selected from a cone-shaped outer segment filled with light-sensitive S-, M- or L-opsin for light signal transduction, a connecting cilium between the inner and outer segments, an inner segment filled with ribosomes and mitochondria for opsin biosynthesis and transport, a cell body containing a nucleus, and an axon with synaptic terminals that allow neurotransmission. Preferably, a cone photoreceptor cell is characterized by two or more morphological features. A morphological feature that is particularly relevant for a person skilled in the art to confirm the generation of an effective cone photoreceptor cell that can detect light is the occurrence of a defined cone-shaped outer segment. As used in the present invention, "cone photoreceptor cell" includes a cone photoreceptor cell or a population of cone photoreceptor cells. Since it is not technically feasible to show in every instance that the "cone photoreceptor cell" generated as described herein exhibits all the characteristics of a "natural" cone photoreceptor cell, the terms "cone photoreceptor cell" and "cone-like photoreceptor cell" are used interchangeably herein.

[0089] As used in this disclosure, "RPE cells" or "RPE-like cells" are any cells that have the characteristics of RPE cells. This may be based on the presence of at least one marker and / or at least one morphological feature and / or at least one functional feature of RPE cells. The morphology of RPE is well described in the literature. The morphology of RPE may include the presence of pigmented melanosomes and / or epithelial phenotype and / or monolayer formation and / or polarization. The at least one marker of RPE cells may be selected from the group consisting of BEST1, MITF, ITGAV, ITGB5, MFGE8, CD81, MERTK, GAS6, PTK2, RLBP1, RPE65, LRAT, RDH5, RDH10, RDH11, PMEL, TYR, TYRP1, GPR143, DCT, OCA2, RAB38 and MYRIP. The at least one marker may be any one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, six, seven, eight, nine, twenty, twenty-one, twenty-two or twenty-three of BEST1, MITF, ITGAV, ITGB5, MFGE8, CD81, MERTK, GAS6, PTK2, RLBP1, RPE65, LRAT, RDH5, RDH10, RDH11, PMEL, TYR, TYRP1, GPR143, DCT, OCA2, RAB38 and MYRIP, including any combination thereof. The at least one marker of RPE cells may be selected from the group consisting of RPE-65, BEST1, MITF and PMEL. At least one marker of RPE cells can be any one, two, three or four of RPE-65, BEST1, MITF and PMEL, including any combination thereof. Thus, RPE-like cells can express RPE-65, BEST1, MITF and / or PMEL. For example, RPE-like cells can express BEST1, MITF and PMEL. At least one morphological feature of RPE cells can be selected from pigment-containing melanosomes, epithelial phenotype, monolayer formation and / or polarization. At least one functional feature of RPE cells can be selected from phagocytosis of cone photoreceptor outer segments; visual cycle (the biochemical process of regenerating chromophores); and pigmentation.Preferably, the RPE-like cells are characterized by two or more functional characteristics of RPE cells. The RPE-like cells can express genes related to one or more functional characteristics of RPE cells. The RPE-like cells can express genes related to the phagocytosis of cone photoreceptor outer segments. The genes related to the phagocytosis of cone photoreceptor outer segments can be selected from the group consisting of ITGAV, ITGB5, MFGE8, CD81, MERTK, GAS6 and PTK2. The genes related to the phagocytosis of cone photoreceptor outer segments can be related to the binding stage of phagocytosis. The genes related to the binding stage of phagocytosis can be selected from the group consisting of ITGAV, ITGB5, MFGE8 and CD81. The genes related to the phagocytosis of cone photoreceptor outer segments can be related to the internalization stage of phagocytosis. The genes related to the internalization stage of phagocytosis can be selected from the group consisting of MERTK, GAS6 and PTK2. The RPE-like cells can express genes related to the visual cycle. The genes related to the visual cycle can be selected from the group consisting of RLBP1, RPE65, LRAT, RDH5, RDH10 and RDH11. The RPE-like cells can express genes related to pigmentation. The genes related to pigmentation can be selected from the group consisting of PMEL, TYR, TYRP1, GPR143, DCT, OCA2, RAB38 and MYRIP. The "RPE cells" used according to the present invention encompass RPE cells or populations of RPE cells. Since it is not technically feasible to show in every instance that the "RPE cells" generated as described herein exhibit all the characteristics of "natural" RPE cells, the terms "RPE cells" and "RPE photoreceptor cells" are used interchangeably herein.

[0090] As used herein, "cell conversion" refers to the method of "transdifferentiation" or "direct cell conversion", which may also be referred to as "forward programming", i.e., changing the morphology and / or phenotype of one terminally differentiated cell, such as the source cell of the present invention, to another terminally differentiated cell. The process of transdifferentiation can be either "direct", which does not go through an intermediate pluripotent state or a precursor cell type, or "indirect", which the process goes through an intermediate pluripotent state or a precursor cell type. The method of inducing "indirect" transdifferentiation can be recognized by a process involving transcription factors associated with the pluripotent phase, for example, the Yamanaka factors OCT3, OCT4 (also known as POU5F1), SOX2, KLF4 and cMYC are known to be highly expressed in embryonic stem (ES) cells. Additional pluripotency transcription factors well known to contribute to the reprogramming of somatic cells to a pluripotent-like state include NANOG, LIN28 and GLIS1. The term "cell conversion" can be used interchangeably with the term "transdifferentiation" or "direct cell conversion". Advantageously, "cell conversion" allows for the conversion of cells derived from one lineage to cells derived from the same or a different lineage. For example, the present invention converts source cells selected from astrocytes or Müller glial cells derived from a neuroectodermal lineage into cone photoreceptors, which are also derived from neuroectodermal.

[0091] Without being bound by theory, the transcription factors disclosed herein can convert macroglial source cells into cone-like photoreceptor cells and / or RPE-like cells via progenitor cells.Since the transcription factors are not pluripotency-inducing, none of these progenitor cells will become pluripotent cells such as iPSCs, but they can be or have one or more characteristics of pluripotent cells, proliferating cells and / or Müller glia progenitor cells.Alternatively, the transcription factors disclosed herein can convert macroglial source cells into cone-like photoreceptor cells and / or RPE-like cells directly.

[0092] The present disclosure provides a method for cellular conversion of Müller glial cells into cone photoreceptor cells by introducing at least one or more transcription factors or variants thereof, or any combination thereof, selected from the group consisting of MEF2C, MEF2D and RXRG, and optionally CRX, into Müller glial cells, thereby generating cone photoreceptors. Müller glial cells are cultured for a sufficient time and under conditions to allow direct conversion into photoreceptor cells, where at least 0.01% of Müller glial cells are converted into photoreceptor cells. In some embodiments, at least 0.01%, at least 0.02%, at least 0.05%, at least 0.075%, or at least 0.1% of Müller glial cells are converted into photoreceptor cells. In other embodiments, at least 0.1%, at least 0.5%, at least 1%, at least 5%, or at least 10% of Müller glial cells are converted into photoreceptor cells. In further embodiments, at least 10%-20%, preferably more than 50%, more preferably at least 85%, 95% or 100% of the Müller glial cells are converted to photoreceptor cells. By "converted" it is meant that there is an upregulation of any one or more cell markers of cone photoreceptors, or a downregulation of one or more cell markers of source cells (e.g., one or more markers of Müller glia or astrocytic cells), and / or one or more changes in cell morphology from source to cone photoreceptors, or any combination thereof. The change in morphology can be identified by observation at the microscopic level. For example, in the case of conversion of Müller glial cells to cone photoreceptors, the morphological changes include a change from elongated cells that traverse all layers of the retina to photoreceptor-like cells with cone outer segments in the ONL. The upregulation of markers can be observed by staining cells for specific markers, or by analyzing the expression of markers at the gene level, or by performing single-cell RNA sequencing.For example, in the case of conversion of Muller glial cells to cone photoreceptors, the upregulation of at least one marker includes upregulation of the specific cone marker, cone arrestin.

[0093] Upregulation of cone cell markers, downregulation of source cell markers, and / or changes in cell morphology are all believed to be characteristics of cone photoreceptor cells generated by the methods of the present invention, hi some embodiments, at least 0.01%, at least 0.05%, at least 0.1%, at least 0.5%, at least 1%, at least 5%, or at least 10% of the converted cone photoreceptors exhibit at least one characteristic of a cone photoreceptor.

[0094] The present disclosure provides a method for cellular conversion of Müller glial cells into RPE-like cells by introducing at least one or more transcription factors, including MEF2C, or their variants into Müller glial cells, thereby generating RPE-like cells. The one or more transcription factors can further include one or more of MEF2D and RXRG. The one or more transcription factors can further include CRX. Müller glial cells are cultured for a sufficient time and under conditions to allow direct conversion into RPE-like cells, where at least 0.01% of Müller glial cells are converted into RPE-like cells. In some embodiments, at least 0.01%, at least 0.02%, at least 0.05%, at least 0.075%, or at least 0.1% of Müller glial cells are converted into RPE-like cells. In other embodiments, at least 0.1%, at least 0.5%, at least 1%, at least 5%, or at least 10% of Müller glial cells are converted into RPE-like cells. In further embodiments, at least 10%-20%, preferably more than 50%, more preferably at least 85%, 95% or 100% of the Müller glial cells are converted to RPE-like cells. By "converted" it is meant that there is an upregulation of any one or more cell markers of RPE-like cells, or a downregulation of one or more cell markers of source cells (e.g., one or more markers of Müller glia or astrocyte cells), and / or one or more changes in cell morphology from source to RPE-like cells, or any combination thereof. The change in morphology can be identified by observing at a microscopic level. For example, in the case of converting Müller glia cells to RPE-like cells, the morphological changes include a change from elongated cells that cross all layers of the retina to pigmented cells. The upregulation of markers can be observed by staining cells for specific markers, or by analyzing the expression of markers at the gene level, or by performing single-cell RNA sequencing.For example, in the case of conversion of Müller glia cells to RPE-like cells, the upregulation of at least one marker includes upregulation of BEST1, a specific RPE cell marker.

[0095] Upregulation of RPE-like cell markers, downregulation of source cell markers, and / or changes in cell morphology are all believed to be characteristics of RPE-like cells generated by the methods of the present invention, hi some embodiments, at least 0.01%, at least 0.05%, at least 0.1%, at least 0.5%, at least 1%, at least 5%, or at least 10% of the converted RPE-like cells exhibit at least one characteristic of RPE-like cells.

[0096] The present disclosure provides a method for converting a source cell selected from a Müller glia or astrocyte cell into a cone photoreceptor cell by introducing one or more nucleic acids, thereby enhancing the expression in the source cell of one or more transcription factors or variants thereof selected from MEF2C, MEF2D and RXRG, or any combination thereof. The one or more transcription factors can include at least one, at least two, or at least three of MEF2C, MEF2D and RXRG in any combination thereof. In one embodiment, the one or more transcription factors can be a single transcription factor, for example; MEF2C or MEF2D or RXRG alone. In other embodiments, the one or more transcription factors can be a combination of two transcription factors in any combination, for example; MEF2C and MEF2D, or MEF2C and RXRG, or MEF2D and RXRG. In other embodiments, the one or more transcription factors can be a combination of three transcription factors, for example; MEF2C, MEF2D and RXRG. In other embodiments, the one or more nucleic acids further encode CRX. In one embodiment, the one or more nucleic acids encode MEF2C, MEF2D, RXRG and CRX, thereby increasing expression of all four transcription factors in the source cell.

[0097] The present disclosure provides a method for converting a source cell selected from a Muller glial cell or an astrocyte into a cone photoreceptor cell, the method comprising: i) providing a source cell or a cell population comprising source cells; ii) introducing into the source cell one or more nucleic acids encoding at least one or more transcription factors or variants thereof selected from the group consisting of MEF2C, MEF2D, RXRG and CRX, thereby enhancing expression of the at least one or more transcription factors; iii) culturing the source cells or a cell population comprising the source cells until at least one characteristic of a cone photoreceptor cell is observed. Includes.

[0098] The present disclosure provides a method for converting a source cell selected from a Muller glial cell or an astrocyte into a cone photoreceptor cell, the method comprising: i) providing a source cell or a cell population comprising source cells; ii) introducing into the source cell one or more nucleic acids encoding at least one or more transcription factors, including MEF2C (and optionally further including MEF2D and / or RXRG and / or CRX), or variants thereof, thereby enhancing expression of the at least one or more transcription factors; iii) culturing the source cells or a cell population comprising the source cells until at least one characteristic of a cone-like photoreceptor cell is observed. Includes.

[0099] The present disclosure provides a method for converting a source cell selected from a Müller glial cell or an astrocyte into an RPE-like cell, the method comprising: i) providing a source cell or a cell population comprising source cells; ii) introducing into the source cell one or more nucleic acids encoding at least one or more transcription factors, including MEF2C (and optionally further including MEF2D and / or RXRG and / or CRX), or variants thereof, thereby enhancing expression of the at least one or more transcription factors; iii) culturing the source cells or a cell population comprising the source cells until at least one characteristic of RPE-like cells is observed. Includes.

[0100] Those skilled in the art will be familiar with culturing cells routinely and therefore using appropriate media and culture conditions that support the development and proliferation of Müller glia cells. Source cells, e.g. Müller glia cells and astrocytes, can be cultured according to standard protocols to generate retinal organoids from iPSC cultures and retinal explant cultures, whereas astrocytes are only present in brain organoids and retinal explants, since they are not derived from retinal progenitor cells like Müller glia. Transduced cells can be monitored until at least one characteristic of cone photoreceptor cells and / or RPE cells is observed, e.g. using fluorescent proteins as part of viral vectors, e.g. GFP. The culture time until at least one characteristic of cone photoreceptor cells and / or RPE cells is observed may vary depending on the experimental system and the choice of vector. For example, less time may be required when repRNA vectors are used, compared to viral vectors such as lentivirus or AAV. Also, less time may be required in 2D Müller glia cultures than in 3D retinal organoids. If the cells are 2D Müller glia and the vector is repRNA, the cells can be cultured for about 2 days to about 7 days (e.g., at least 2 days, at least 3 days, at least 4 days, at least 5 days, or at least 6 days). If the cells are 2D Müller glia and the vector is a viral vector such as lentivirus or AAV, the cells can be cultured for about 7 days to about 14 days (e.g., at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, or at least 13 days).If the cells are retinal organoids, the cells can be cultured for about 7 days to about 28 days (e.g., at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, at least 14 days, at least 15 days, at least 16 days, at least 17 days, at least 18 days, at least 19 days, at least 20 days, at least 21 days, at least 22 days, at least 23 days, at least 24 days, at least 25 days, at least 26 days, or at least 27 days). If the cells are retinal explants, such as mouse retinal explants, the cells can be cultured for about 10 days to about 14 days (e.g., at least 10 days, at least 11 days, at least 12 days, or at least 13 days). Appropriate media for culturing source cells, such as Müller glia cells and astrocytes, are known to those skilled in the art. Examples of suitable media include B27-based and N2-based retinal differentiation media. Once retinal organoids have been generated from iPSCs, the source cells may be cultured for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59 or 60 days, preferably the source cells are cultured for between 14 and 21 days after transduction. The length of culture time may be the entire culture time, i.e., before transducing the cells and until the cells have transdifferentiated into cone photoreceptors, or the length of culture time may be the culture time after transduction of the source cells.

[0101] Small molecules can be used to activate Müller glia cells, induce GFAP expression, and thus enhance their proliferation. An example of a small molecule that can enhance the activation and proliferation of Müller glia cells is purmorphamine. Activation of Müller glia is required in human retinal organoids. Because Müller glia are "healthy" and do not show GFAP expression when they are developing and not in disease state. Purmorphamine is not required to treat patients with retinal degeneration. Because Müller glia are activated and express GFAP in retinal disease and retinal trauma. Thus, the method may include contacting cells with purmorphamine. Thus, the method may include contacting cells with purmorphamine, where one or more transcription factors are under the control of GFAP promoter.

[0102] As used herein, "transcription factor" refers to a protein whose function is to regulate the expression of a particular gene or genes in a cell, controlling the rate of transcription of the DNA of said genes into messenger RNA.

[0103] As used herein, "MEF2C" refers to myocyte enhancer factor 2C. MEF2C is also known as myocyte-specific enhancer factor 2D or MADS-box transcriptional enhancer factor 2, polypeptide C. MEF2C belongs to the MEF2 family of transcription factors that play a role in myogenesis. MEF2A is known to be an important paralog of the MEF2C gene. Ensembl gene ID is ENSG00000081189.16. An example of a transcript is Ensembl transcript ID ENST00000636998.1, UniParc ID Q06413-3, and includes transcript variants due to codon optimization and alternative splicing. MEF2C is a transcription factor that has been shown to directly reprogram cardiac fibroblasts into cardiomyocytes.

[0104] MEF2C can have the following amino acid sequence, identified as SEQ ID NO:1. SEQ ID NO:1 MGRKKIQITRIMDERNRQVTFTKRKFGLMKKAYELSVLCDCEIALIIFNSTNKLFQYASTDMDKVLLKYTEYNEPHESRTNSDIVETLRKKGLNGCDSPDPDADDSVGHS PESEDKYRKINEDIDLMISRQRLCAVPPPNFEMPVSIPVSSHNSLVYSNPVSSLGNPNLLPLAHPSLQRNSMSPGVTHRPPSAGNTGGLMGGDLTSGAGTSAGNGYGNPR NSPGLLVSPGNLNKNMQAKSPPPMNLGMNNRKPDLRVLIPPGSKNTMPSVSEDVDLLLNQRINNSQSAQSLATPVVSVATPTLPGQGMGGYPSAISTTYGTEYSLSSADL SSLSGFNTASALHLGSVTGWQQQHLHNMPPSALSQLGDRTTTPSRYPQHTRHEAGRSPVDSLSSCSSSYDGSDREDHRNEFHSPIGLTRPSPDERESPSVKRMRLSEGWAT MEF2C can be encoded by the following "wild type" sequence, identified as SEQ ID NO:2. SEQ ID NO:2 MEF2C can be encoded by the following "codon optimized" sequence identified as SEQ ID NO:3. SEQ ID NO:3 As used herein, "MEF2D" refers to myocyte enhancer factor 2D. MEF2D is also known as myocyte specific enhancer factor 2D or MADS box transcription enhancer factor 2, polypeptide D. MEF2D belongs to the MEF2 family of transcription factors. MEF2A is known to be an important paralog of the MEF2D gene. Ensembl gene ID is ENSG00000116604.19. An example of a transcript is Ensembl transcript ID ENST00000348159.9, UniParc ID Q14814-1, and includes transcript variants due to codon optimization and alternative splicing. MEF2D is a transcription factor, which is known to mediate cell functions in skeletal and cardiac muscle development, as well as neuronal differentiation and survival, and to play a pivotal role in regulating neuronal apoptosis.

[0105] The MEF2D may have the following amino acid sequence, identified as SEQ ID NO:4. SEQ ID NO:4 MGRKKIQIQRITDERNRQVTFTKRKFGLMKKAYELSVLCDCEIALIIFNHSNKLFQYASTDMDKVLLKYTEYNEPHESRTNADIIETLRKKGFNGCDSPEPDGEDSLEQSPLLEDKYRRASEELDGLFRR YGSTVPAPNFAMPVTVPVSNQSSLQFSNPSGSLVTPSLVTSSLTDPRLLSPQQPALQRNSVSPGLPQRPASAGAMLGGDLNSANGACPSPVGNGYVSARASPGLLPVANGNSLNKVIPAKSPPPPTHSTQ LGAPSRKPDLRVITSQAGKGLMHHLTEDHLDLNNAQRLGVSQSTHSLTTPVVSVATPSLLSQGLPFSSMPTAYNTDYQLTSAELSSLPAFSSPGGLSLGNVTAWQQPQQPQQPQQPQPPQQQPPQPQQPQ PQQPQQPQQPPQQQSHLVPVSLSNLIPGSPLPHVGAALTVTTHPHISIKSEPVSPSRERSPAPPPPAVFPAARPEPGDGLSSPAGGSYETGDRDDGRGDFGPTLGLLRPAPEPEAEGSAVKRMRLDTWTLK MEF2D can be encoded by the following "wild type" sequence, identified as SEQ ID NO:5. SEQ ID NO:5 MEF2D can be encoded by the following "codon optimized" sequence identified as SEQ ID NO:6. SEQ ID NO:6 As used herein, "RXRG" refers to retinoid X receptor gamma. RXRG is also known as retinoic acid receptor RXR-gamma, retinoid X receptor gamma, NR2B3, and nuclear receptor subfamily 2 group B member 3. Ensembl gene ID is ENSG00000143171.13. An example transcript is Ensembl transcript ID ENST00000359842.10 and UniParc ID P48443-1, encompassing transcript variants with codon optimization and alternative splicing. RXRG is a transcription factor that belongs to the retinoid X receptor (RXR) family of nuclear receptors and is known to be involved in mediating the anti-proliferative effects of retinoic acid (RA). There are three subtypes of RXR genes, alpha (RXRA), beta (RXRB) and gamma (RXRG), and RXRA is known to be an important paralog of RXRG.

[0106] RXRG can have the following amino acid sequence, identified as SEQ ID NO:7. SEQ ID NO:7 MYGNYSHFMKFPAGYGGSPGHTGSTSMSPSAALSTGKPMDSHPSYTDTPVSAPRTLSAVGTPLNALGSPYRVITSAMGPPSGALAAPPGINLVAPPSSQLNVVNSVSSSEDIKPL PGLPGIGNMNYPSTSPGSLVKHICAICGDRSSGKHYGVYSCEGCKGFFKRTIRKDLIYTCRDNKDCLIDKRQRNRCQYCRYQKCLVMGMKREAVQEERQRSRERAESEAECATSGH EDMPVERILEAELAVEPKTESYGDMNMENSTNDPVTNICHAADKQLFTLVEWAKRIPHFSDLTLEDQVILLRAGWNELLIASFSHRSVSVQDGILLATGLHVHRSSAHSAGVGSIF DRVLTELVSKMKDMQMDKSELGCLRAIVLFNPDAKGLSNPSEVETLREKVYATLEAYTKQKYPEQPGRFAKLLLRLPALRSIGLKCLEHLFFFKLIGDTPIDTFLMMEMLETPLQIT RXRG can be encoded by the following "wild type" sequence, identified as SEQ ID NO:8. SEQ ID NO:8 RXRG can be encoded by the following "codon-optimized" sequence, identified as SEQ ID NO:9. SEQ ID NO:9 As used herein, "CRX" refers to Cone-Rod Homeobox. CRX is also known as CORD2, LCA7, OTX3, CRD or Orthodenticle Homeobox 3. CRX is considered to be part of the OTX family of genes. Ensembl gene ID is ENSG00000105392.16. An example of a transcript is Ensembl transcript ID ENST00000221996.12 and UniParc ID O43186-1, which includes transcript variants with codon optimization and alternative splicing. The protein encoded by the CRX gene is a photoreceptor-specific transcription factor, which plays a role in the differentiation of photoreceptor cells and is necessary for the maintenance of normal cone and rod function. The CRX gene acts synergistically with other transcription factors such as NRL, RORB and RAX to regulate the transcription of photoreceptor-specific genes. OTX2 is known to be an important paralog of the CRX gene and can be used interchangeably with CRX as a transcription factor according to the present invention.

[0107] CRX may have the following amino acid sequence, identified as SEQ ID NO:10. SEQ ID NO:10 MMAYMNPGPHYSVNALALSGPSVDLMHQAVPYPSAPRKQRRERTTFTRSQLEELEALFAKTQYPDVYAREEVALKINLPESRVQVWFKNRRAKCRQQRQQQKQQQQPPGGQAKARPAKRKAGTSPRPSTDVCPDPLGISDSYSPPLPGP SGSPTTAVATVSIWSPASESPLPEAQRAGLVASGPSLTSAPYAMTYAPASAFCSSPSAYGSPSSYFSGLDPYLSPMVPQLGGPALSPLSGPSVGPSLAQSPTSLSGQSYGAYSPVDSLEFKDPTGTWKFTYNPMDPLDYKDQSAWKFQIL CRX may be encoded by the following "wild type" sequence identified as SEQ ID NO:11. SEQ ID NO:11 ATGATGGCGTATATGAACCCGGGGCCCCACTATTCTGTCAACGCCTTGGCCCTAAGTGGCCCCAGTGTGGATCTGATGCACCAGGCTGTGCCCTACCCAAGCGCCCCCAGGAAGCAGCGGCGGGAGCGCACCACCTTCACCCGGAGCCAACTGGAGGAGCTGGAGGCACTGTTTGCCAAGACCCAGTACCCAGACGTCTATGCCCGTGAGGAGGTGGCTCTGAAGATCAATCTGCCTGAGTCCAGGGTTCAGGTTTGGTTCAAGAACCGGAGGGCTAAATGCAGGCAGCAGCGACAGCAGCAGAAACAGCAGCAGCAGCCCCCAGGGGGCCAGGCCAAGGCCCGGCCTGCCAAGAGGAAGGCGGGCACGTCCCCAAGACCCTCCACAGATGTGTGTCCAGACCCTCTGGGCATCTCAGATTCCTACAGTCCCCCTCTGCCCGGCCCCTCAGGCTCCCCAACCACGGCAGTGGCCACTGTGTCCATCTGGAGCCCAGCCTCAGAGTCCCCTTTGCCTGAGGCGCAGCGGGCTGGGCTGGTGGCCTCAGGGCCGTCTCTGACCTCCGCCCCCTATGCCATGACCTACGCCCCGGCCTCCGCTTTCTGCTCTTCCCCCTCCGCCTATGGGTCTCCGAGCTCCTATTTCAGCGGCCTAGACCCCTACCTTTCTCCCATGGTGCCCCAGCTAGGGGGCCCGGCTCTTAGCCCCCTCTCTGGCCCCTCCGTGGGACCTTCCCTGGCCCAGTCCCCCACCTCCCTATCAGGCCAGAGCTATGGCGCCTACAGCCCCGTGGATAGCTTGGAATTCAAGGACCCCACGGGCACCTGGAAATTCACCTACAATCCCATGGACCCTCTGGACTACAAGGATCAGAGTGCCTGGAAGTTTCAGATCTTGTAG CRX can be encoded by the following "codon-optimized" sequence, specified as SEQ ID NO: 12. SEQ ID NO: 12 atgatggcctacatgaatcctggtccacattactcagtaaatgctctcgcacttagcggtcctagcgtagacctcatgcatcaagcggtcccgtatccctcagcgccgcggaaacaaagacgtgaaaggacaacatttactagatctcagcttgaagaactcgaagccttgttcgcgaaaacacaatatcccgatgtgtacgcacgggaagaagtcgccctcaaaataaacttgccagaatctcgtgtacaagtgtggtttaagaatagacgcgccaagtgtagacaacaaagacaacaacaaaagcaacaacaacaaccgccgggcggtcaagctaaagctaggcccgctaaacggaaagctggtacaagcccccggccgtcaactgacgtttgccccgatccacttggtatttctgactcttattccccgcccttgcctggaccgagcggtagtcccacaaccgctgtcgcaacggtatctatttggtccccggccagcgaatctcccctgccagaagcacaacgcgcaggcctcgtcgcatccggcccttcactcactagcgcaccatacgcaatgacgtatgcaccagctagtgcattttgttcctcaccttctgcatacggcagcccctcatcatacttttctgggctggacccttatctgtcacctatggtcccgcaattgggcggacctgccctgagtccacttagcggaccgagcgtcggtccgtcactcgctcaaagcccgacatctctcagcggtcaatcttacggagcatattcacctgtcgactcccttgagtttaaagatccaactgggacatggaagtttacttataacccaatggacccactcgattataaagaccaatccgcgtggaaattccaaattctctga As used herein, "OTX2" refers to Orthodenticle Homeobox 2. OTX2 is also considered to be part of the OTX family of genes. The Ensembl gene ID is ENSG00000165588.19. An example transcript is Ensembl transcript ID ENST00000672264.2 and UniParc ID P32243-1, encompassing transcript variants due to codon optimization and alternative splicing. The protein encoded by the OTX2 gene is a transcription factor, which plays a broad role in the development and function of cone photoreceptor cells. The single Drosophila orthodenticle (otd) gene is represented by three vertebrate OTX factors, OTX1, OTX2 and CRX. OTX2 is well established as a key regulator of retinal development and is essential for retinal pigment epithelium and photoreceptor differentiation, while OTX1 may be important for ciliary body development. CRX is a photoreceptor-specific gene known to be essential for photoreceptor development.

[0108] "Engineered" or "non-naturally occurring" are terms that indicate human involvement. When referring to a nucleic acid molecule or polypeptide, the term indicates that the nucleic acid molecule or polypeptide is at least substantially free of at least one other component with which it naturally associates and is found in its natural state. In addition, the term may indicate that the nucleic acid molecule or polypeptide has a sequence that is not found in the natural state.

[0109] The term "enhancing or enhancing" as used herein means to induce or enhance or activate the level, activity or function or expression of at least one or more transcription factors in a source cell. Preferably, the activity is induced or enhanced or activated by 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more compared to a comparison value (i.e., a control). For example, the control may be a source cell into which a nucleic acid encoding expression cassette has not been introduced. In some embodiments, the amount of the transcription factor is enhanced in the cell (e.g., via an expression cassette that induces expression of a nucleic acid molecule encoding one or more transcription factors) compared to a control (e.g., a source cell without said expression cassette). In further embodiments, "enhancing or enhancing" as used herein means to enhance the amount of at least one or more transcription factor proteins in a source cell. In another further embodiment, enhancing expression includes "overexpressing" the transcription factor, i.e., enhancing expression of the transcription factor above the endogenous expression level of the transcription factor in the cell.

[0110] "Sequence identity" refers to the similarity between amino acid (or nucleotide) sequences. Sequence identity is often measured in terms of percentage of identity (or similarity or homology); the higher the percentage, the more similar the two sequences are. Although there are several methods to measure the identity between two polypeptide sequences or two polynucleotide sequences, commonly used methods to determine identity are codified in computer programs. Preferred computer programs to determine identity between two sequences include, but are not limited to, the GCG program package (Devereux, et al., Nucleic Acids Research, 12, 387 (1984)), BLASTP, BLASTN and FASTA (Atschu et al., J. Molec. Biol. (1990) 215, 403). Preferably, a sequence has at least 70% identity with a sequence of the present disclosure using the default parameters of the BLAST computer program (Atschul et al., J. Mol. Biol. (1990) 215, 403-410) provided by HGMP (Human Genome Mapping Project). The term "variant" in reference to protein and nucleic acid sequences (including the MEF2C, MEF2D, RXRG and CRX sequences provided herein) is typically characterized by the possession of at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity counted over a full-length alignment with an amino acid or nucleotide sequence. The present invention contemplates the use of variants of the transcription factors described herein. Variants could be fragments of the full-length sequence, codon-optimized sequences, or naturally occurring splice variants.A variant may be a polypeptide or nucleic acid molecule that is at least 70%, 80%, 85%, 90%, 95%, 98%, or 99% identical to the full-length sequence, where the fragment is at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 98%, or 99%, as long as the full-length wild-type polypeptide or nucleic acid molecule, or a domain thereof, has the desired functional activity, such as the ability to promote the conversion of a source cell type to a target cell type. In some embodiments, the domain is at least 100, 200, 300, or 400 amino acids in length, starting at any amino acid position in the sequence and extending toward the C-terminus. Variations known in the art that eliminate or substantially reduce the activity of a protein are preferably avoided. In some embodiments, the variant lacks the N- and / or C-terminal portion of the full-length polypeptide, for example, lacking up to 10, 20, or 50 amino acids from either end. In some embodiments, a polypeptide has the sequence of a mature (full-length) polypeptide, meaning that it has had one or more portions, such as a signal peptide, removed during normal intracellular proteolytic processing (e.g., during co-translational or post-translational processing). In some embodiments, if a protein is produced other than by purifying it from a cell that naturally expresses it, it is a chimeric polypeptide, meaning that the chimeric polypeptide contains portions from two or more different species. In some embodiments, if a protein is produced other than by purifying it from a cell that naturally expresses it, it is a derivative, meaning that the protein contains additional sequences not associated with the protein, so long as the additional sequences do not substantially reduce the biological activity of the protein. Those skilled in the art will know, or will be able to easily ascertain, whether a particular polypeptide variant, fragment, or derivative is functional using assays known in the art.Other convenient assays include measuring the ability to activate transcription of a reporter construct that contains a transcription factor binding site operably linked to a nucleic acid sequence that codes for a detectable marker. For example, a person skilled in the art knows how to perform a luciferase assay to determine whether a transcription factor variant is a functional variant. For example, if a variant activates a luciferase assay and / or has at least 50% of the activity of a full-length wild-type polypeptide molecule or nucleic acid molecule, the variant can be defined as a functional variant. Alternatively, a person skilled in the art can compare the transcriptome changes upon exposure to a transcription factor and a transcription factor variant, for example using RNAseq, to ​​determine whether a transcription factor variant is a functional variant. The transcriptome changes can be up-regulation and / or down-regulation of gene expression. For example, if a variant up-regulates at least 50% of the genes that are up-regulated by a full-length wild-type polypeptide molecule or nucleic acid molecule, the variant can be defined as a functional variant. Alternatively, a variant may be defined as a functional variant if it downregulates at least 50% of the genes that are downregulated by the full-length wild-type polypeptide or nucleic acid molecule. Alternatively, a variant may be defined as a functional variant if it upregulates at least 50% of the genes that are upregulated by the full-length wild-type polypeptide or nucleic acid molecule and downregulates at least 50% of the genes that are downregulated by the full-length wild-type polypeptide or nucleic acid molecule.Alternatively, to determine whether a transcription factor variant is a functional variant, one skilled in the art can compare the effect of the transcription factor and the transcription factor variant on cell conversion, for example, by comparing the effect on the reduction of one or more characteristics of the source cell (such as Müller glia cells) and / or the increase of one or more characteristics of the target cell (such as cone photoreceptor cells and / or RPE cells). For example, if at least 50% of the cells that downregulate one or more source cell characteristics (e.g., the presence of at least one marker and / or at least one morphological feature of Müller glia cells) upon exposure to a full-length wild-type polypeptide molecule or nucleic acid molecule downregulate one or more source cell characteristics upon exposure to a variant transcription factor, the variant can be defined as a functional variant. Alternatively, a variant may be defined as a functional variant if at least 50% of the cells that upregulate one or more target cellular features (e.g., the presence of at least one marker and / or at least one morphological feature of cone photoreceptor cells and / or the presence of at least one marker and / or at least one morphological feature and / or at least one functional feature of RPE cells) upon exposure to a full-length wild-type polypeptide molecule or nucleic acid molecule also upregulate one or more target cellular features upon exposure to a variant transcription factor. Those skilled in the art know how to carry out an assay of BDNF expression or secretion to determine whether a transcription factor variant is a functional variant. For example, a variant may be defined as a functional variant if it has at least 50% of the BDNF expression of a full-length wild-type polypeptide molecule or nucleic acid molecule. Alternatively, a variant may be defined as a functional variant if it has at least 50% of the BDNF secretion of a full-length wild-type polypeptide molecule or nucleic acid molecule.In certain embodiments of the present invention, functional variants or fragments have at least 50%, 60%, 70%, 80%, 90%, 95% or more of the activity of the full-length wild-type polypeptide or nucleic acid molecule. In certain embodiments of the present invention, functional variants or fragments have at least 50%, 60%, 70%, 80%, 90%, 95% or more of the activity of the codon-optimized molecule disclosed herein; the reference to the full-length wild-type polypeptide or nucleic acid molecule in the above description of how to determine whether a particular variant, fragment or derivative is functional may be replaced with a reference to the codon-optimized molecule disclosed herein. As used herein, the term "functional variant" preferably refers to a molecule, e.g., a polypeptide or nucleic acid molecule, that retains at least about 70% or more (including at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100%) of the biological activity of the wild-type or codon-optimized molecule disclosed herein. Biological activity can refer to, for example, one or more of the following: activity in luciferase assay, changes in transcriptome, cell transformation and / or effects on BDNF expression or secretion.Preferably, "functional variant" is defined by reference to both sequence identity percentage and biological activity.For example, "functional variant" can retain at least 70% (including at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity counted over full length alignment with amino acid or nucleotide sequence), and at least about 70% or more (including at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100%) of biological activity of wild-type or codon-optimized molecules disclosed herein.A "functional variant" can retain at least 80% (including at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity counted over a full-length alignment with an amino acid or nucleotide sequence) and at least about 80% or more (including at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100%) of the biological activity of a wild-type or codon-optimized molecule disclosed herein. A "functional variant" can retain at least 90% (including at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity counted over a full-length alignment with an amino acid or nucleotide sequence) and at least about 90% or more (including at least 95%, at least 97%, at least 99%, or 100%) of the biological activity of a wild-type or codon-optimized molecule disclosed herein. A "functional variant" can retain at least 95% (including at least 96%, at least 97%, at least 98%, or at least 99% sequence identity counted over a full-length alignment with an amino acid or nucleotide sequence) and at least about 95% or more (including at least 97%, at least 99%, or 100%) of the biological activity of a wild-type or codon-optimized molecule disclosed herein.

[0111] The variants may include one or more amino acid substitutions. Amino acid substitutions are usually conservative, i.e., replacement of one amino acid with another amino acid with similar properties, so that the overall function is probably not significantly affected. Thus, the amino acids glycine, alanine, valine, leucine, and isoleucine may often be substituted for each other (amino acids with aliphatic side chains). Of these possible substitutions, glycine and alanine are used to replace each other (because they have relatively short side chains), and valine, leucine, and isoleucine are used to replace each other. They are preferably used because they have larger aliphatic side chains that are hydrophobic. Other amino acids that may often be substituted for each other include phenylalanine, tyrosine, and tryptophan (amino acids with aromatic side chains); lysine, arginine, and histidine (amino acids with basic side chains); aspartic acid and glutamic acid (acidic side chains); asparagine and glutamine (amino acids with amide side chains); and cysteine ​​and methionine (amino acids with sulfur-containing side chains).

[0112] The term "exogenous" when used in reference to a protein, gene, nucleic acid or polynucleotide in a cell or organism refers to a protein, gene, nucleic acid or polynucleotide that has been introduced into the cell or organism by artificial or natural means; or in reference to a cell, refers to a cell that has been isolated and subsequently introduced into another cell or organism by artificial or natural means. An exogenous nucleic acid may be from a different organism or cell, or an exogenous nucleic acid may be one or more additional copies of a nucleic acid that occurs naturally in an organism or cell. An exogenous cell may be from a different organism, or an exogenous cell may be from the same organism. As a non-limiting example, an exogenous nucleic acid is one that is at a chromosomal location that is different from the chromosomal location of a natural cell, or is otherwise flanked by a nucleic acid sequence that is different from the nucleic acid sequence found in nature. An exogenous nucleic acid may be extrachromosomal, such as an episomal vector.

[0113] The term "expression cassette" is meant to include any type of genetic construct containing a nucleic acid encoding a gene product, where part or all of the nucleic acid coding sequence can be transcribed and translated, i.e., under the control of a promoter. An "exogenous expression cassette" originates from outside an organism and is exogenously introduced. As used herein, the term "nucleic acid" encompasses DNA or RNA. As used herein, "nucleic acid" includes, but is not limited to, polynucleotides, proteins, oligonucleotides, synthetic mRNA molecules, synthetic RNA molecules (e.g., repRNA), synthetic DNA molecules, siRNAs, RNAi, peptide nucleic acids (PNAs), polypeptides, and peptides.

[0114] Nucleic acids can be delivered by vectors such as viral or non-viral vectors. Viral vectors can be those that integrate into the host cell genome or those that do not, such as, but not limited to, lentivirus, Sendai vector, herpes simplex virus (HSV), adenovirus, adeno-associated virus (AAV), episomal vectors (e.g., EBV vectors) and retroviruses. In particular, AAV capsids can be engineered to enhance gene delivery. Capsid variants can be selected to drive protein expression in source cells, for example, capsid ShH10 from AAV6 has been shown to transduce primarily Müller glia cells in the rat retina, while AAV2.7m8 has a high level of retinal tropism as well (Khabou et al., 2016 Biotechnol. Bioeng. 2016;113:2712-2724). Other capsid variants that have been shown to transduce Müller glia are AAV1, 2, 4, 6, 8, 9, ShHIOY and ShH13 (Koerber, 2009 DOI:10.1038 / mt.2009.184; O'Carroll, 2020 DOI:10.3389 / fnmol.2020.618020). The viral vector can comprise a capsid that exhibits retinal tropism. The viral vector can comprise a capsid for transduction of macroglia, preferably Müller glia. Thus, the viral vector can comprise a capsid selected from the group consisting of AAV1, 2, 4, 6, 8, 9, AAV2.7m8, ShH10, ShH10Y and ShH13. The viral vector can comprise an AAV2 capsid or an AAV2 variant capsid. The viral vector can include AAV6 capsid or AAV6 variant capsid. The AAV capsid can provide specificity for targeting to Muller glia, which can also be performed / enhanced by using Muller glia specific promoters. The vector may or may not integrate into the genome of the cell. If desired, the construct can include viral sequences for transduction.Non-viral vectors include, but are not limited to, liposomes, nanoparticles, naked DNA, plasmids, transposons, and other means of delivering nucleic acid molecules to cells. Another type of vector includes RNA molecules, such as mRNA and stabilized RNA, which carry coding genetic information to cells. Other modalities suitable for facilitating the transfer of nucleic acid to cells can also be used. The terms "vector" and "expression vector" are used interchangeably and refer to vectors that allow the expression of nucleic acid encoding transcription factors. In some embodiments, the gene of interest is operably linked to another sequence in the vector.

[0115] The nucleic acid encoding the transcription factor can be cloned into a monocistronic expression vector, a bicistronic expression vector or a polycistronic expression vector. A monocistronic vector encodes a single transcription factor, a bicistronic expression vector encodes two transcription factors, and a polycistronic expression vector encodes two or more transcription factors. Preferably, in the bicistronic expression vector or the polycistronic expression vector, the nucleic acid encoding the transcription factor is separated by a P2A, T2A, E2A or F2A linker that induces ribosome skipping when the protein is translated, resulting in the production of two or more separate proteins. The nucleic acid encoding the transcription factor is exogenously introduced into the cell.

[0116] "Self-replicating RNA" or "self-amplifying RNA" or "repRNA" is RNA that can be continuously replicated in host cells as a replicon without the need for a DNA template, or can be transcribed by itself. Thus, the repRNA for inducing Muller glial cells to transdifferentiate into cone photoreceptor cells can include a 5' cap, and sequences encoding nonstructural proteins, at least one or more of the MEF2C, MEF2D and RXRG genes, an independent ribosome entry site (IRES), optionally a fluorescent marker gene (GFP or mCherry), and a 3' polyA tail. The repRNA can further include a CRX gene. The repRNA can include a promoter operably linked to a nucleic acid sequence encoding myocyte enhancer factor 2C (MEF2C) or a functional variant thereof, where the promoter is for expression of MEF2C in macroglia, and optionally, the repRNA can include a promoter operably linked to a nucleic acid sequence encoding one or more transcription factors selected from the group consisting of myocyte enhancer factor 2D (MEF2D), retinoid X receptor gamma (RXRG) and cone-rod homeobox (CRX), or functional variants thereof, or any combination thereof, where the promoter is for expression of the one or more transcription factors in macroglia. Additionally, the repRNA molecule can include two RNA molecules: a first molecule that includes a sequence encoding a nonstructural protein required for RNA replication, and a second molecule that includes a sequence encoding at least one transcription factor.

[0117] A "promoter" refers to a DNA sequence that is recognized by the synthetic machinery of a cell or introduced synthetic machinery required to initiate specific transcription of a gene. The phrases "under transcriptional control" or "operably linked to" mean that the promoter is in the correct location and orientation relative to the nucleic acid to control the initiation of RNA polymerase and expression of the gene. A promoter also includes distal enhancer or repressor elements as required. A "constitutive promoter" is a promoter that is continuously active and is not subject to regulation by external signals or molecules. For example, constitutive promoters include CMV and CAG. In contrast, the activity of an "inducible promoter" is regulated by an external signal or molecule (e.g., a transcription factor). A "cell-specific promoter" or "tissue-specific promoter" is a promoter that induces expression of a nucleic acid molecule in a particular cell or tissue; for example, cell-specific promoters known to direct expression in Müller glial cells and astrocytes include retinaldehyde-binding protein 1 (RLBP1) and glial fibrillary acidic protein (GFAP). If a promoter preferentially induces the expression of a nucleic acid molecule in a particular cell or tissue, but also induces expression at a lower level in other cells or tissues, the promoter can be called a "cell-selective promoter" or a "tissue-selective promoter". Some promoters can be used to induce the expression of the nucleic acid that is operably linked to the promoter sequence. Some other promoters that have been shown to cause Müller glia-specific expression are ProB2 and GLAST. Promoters suitable for inducing expression in Müller glia include sequences from nine Müller cell-associated genes: CAR2, CD44, GFAP, GLUL, PDGFRA, RLBP1, S100B, SLC1A3 and vimentin (VIM) and their variants, which have been shown by Geller et al. (2008) Molecular Vision 2008;14:691-705, which is incorporated herein by reference, to be capable of driving reporter gene expression in Müller glia.Promoters described herein with reference to the relevant genes include "full-length" promoters as well as variants of the corresponding promoters. A "full-length" promoter can include approximately 2000 or approximately 1500 bases upstream of the transcription start site (TSS). A variant of a promoter can be a truncated promoter consisting of up to approximately 1000 bases, up to approximately 900 bases, up to approximately 800 bases, up to approximately 700 bases, up to approximately 600 bases, or up to approximately 500 bases of the "full-length" promoter. The bases of the truncated promoter can be consecutive bases proximal to the TSS. "Proximal to" can refer to the base furthest from the TSS being up to 100 bases, up to 90 bases, up to 80 bases, up to 70 bases, up to 60 bases, up to 50 bases, up to 40 bases, up to 30 bases, up to 20 bases, up to 10 bases, up to 5 bases, or 0 bases upstream from the TSS. The TSS may be identified using RefSeq sequences (NCBI), e.g. those compiled in the respective data files available in ECRbase. The promoter variant may be an evolutionarily conserved region (ECR), preferably the ECR most proximal to the TSS. The ECR sequence may be identified within the full-length promoter region, which ensures a minimum of 70% sequence homology between the human and mouse genomes over a window of 100 base pairs. The promoter or variant thereof may further comprise the 5'UTR or a fragment thereof associated with the gene. Preferably, the nucleic acid or nucleic acids encoding at least one or more transcription factors are under the control of the GFAP promoter. The use of the GFAP promoter restricts the expression of the transcription factor to only Müller glia cells and astrocytes. Preferably, a promoter between 400 bp and 800 bp is used, for example the truncated GFAP promoter gfaABC1D having 681 bp (Lee, 2008 DOI:10.1002 / glia.20622), identified below as SEQ ID NO: 13, which is reported to have essentially the same expression pattern as the 2210 bp gfa2 promoter and approximately twice as much activity.

[0118] The GFAP promoter may be a shortened or truncated GFAP promoter. The GFAP promoter may consist of or include SEQ ID NO: 13 (681 bp sequence). The GFAP promoter may consist of or include SEQ ID NO: 14 (699 bp sequence including the sequence of SEQ ID NO: 13). SEQ ID NO:13 AACATATCCTGGTGTGGAGTAGGGGACGCTGCTCTGACAGAGGCTCGGGGGCCTGAGCTGGCTCTGTGAGCTGGGGAGGAGGCAGACAGCCAGGCCTTGTCTGCAAGCAGACCTGGCAGCATTGGGCTGGCCGCCCCCCAGGGCCTCCTCTTCATGCCCAGTGAATGACT CACCTTGGCACAGACACAATGTTCGGGGTGGGCACAGTGCCTGCTTCCCGCCGCACCCCAGCCCCTCAAATGCCTTCCGAGAAGCCCATTGAGCAGGGGGCTTGCATTGCACCCCAGCCTGACAGCCTGGCATCTTGGGATAAAAGCAGCACAGCCCCCTAGGGCTG CCCTTGCTGTGTGGCGCCACCGGCGGTGGAGAACAAGGCTCTATTCAGCCTGTGCCCAGGAAAGGGGATCAGGGGATGCCCAGGCATGGACAGTGGGTGGCAGGGGGGGAGAGGAGGGCTGTCTGCTTCCCAGAAGTCCAAGGACACAAATGGGTGAGGGGAGAGCTCTC CCCATAGCTGGGCTGCGGCCCAACCCCCCCCCTCAGGCTATGCCAGGGGGTGTTGCCAGGGGCACCCGGGCATCGCCAGTCTAGCCCACTCCTTCATAAAGCCCTCGCATCCCAGGAGCGAGCAGAGCCAGAGCAGGTTGGAGAGGAGACGCATCACCTCCGCTGCTCGC SEQ ID NO:14 ctagtgatctaacatatcctggtgtggagtaggggacgctgctctgacagaggctcgggggcctgagctggctctgtgagctggggaggaggcagacagccaggccttgtctgcaagcagacctggcagcattgggctggccgccccccagggcctcctcttcatgcccagtgaatgactcaccttggcacagacacaatgttcggggtgggcacagtgcctgcttcccgccgcaccccagcccccctcaaatgccttccgagaagcccattgagcagggggcttgcattgcaccccagcctgacagcctggcatcttgggataaaagcagcacagccccctaggggctgcccttgctgtgtggcgccaccggcggtggagaacaaggctctattcagcctgtgcccaggaaaggggatcaggggatgcccaggcatggacagtgggtggcagggggggagaggagggctgtctgcttcccagaagtccaaggacacaaatgggtgaggggagagctctccccatagctgggctgcggcccaaccccaccccctcaggctatgccagggggtgttgccaggggcacccgggcatcgccagtctagcccactccttcataaagccctcgcatcccaggagcgagcagagccagagcaggttggagaggagacgcatcacctccgctgctcgcggggatcc As used herein, a promoter "for" the expression of a gene in macroglia refers to a promoter suitable for the expression of a gene in macroglia. To be suitable for the expression of a gene in macroglia, the promoter may be a macroglia-specific promoter or a macroglia-selective promoter. Alternatively, to be suitable for the expression of a gene in macroglia, the promoter may be an inducible promoter or a constitutive promoter. A promoter that is specific to a cell type other than macroglia, particularly a promoter that is specific to a retinal cell type other than retinal macroglia, is not suitable for the expression of a gene in macroglia. The promoter may not be a promoter that is specific to RPE cells (e.g., VMD2 promoter or REG-RPE promoter). The promoter may not be a promoter that is specific to neuronal cells (e.g., human synapsin 1 (hSyn1), neuron-specific enolase (NSE) or calcium / calmodulin-dependent protein kinase IIA (CaMKIIα)). In some embodiments, the promoter is suitable for the selective expression of a gene in macroglia. Promoters that are selective for cell types other than macroglia, particularly retinal cell types other than retinal macroglia, are not suitable for selective expression of genes in macroglia.Thus, the promoter can drive a greater level (e.g., at least 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30 or 50-fold increase) of expression of genes in retinal macroglia compared to the expression level in other retinal cell types, such as RPE cells, or neuronal cell types, such as cones, rods or RGCs.

[0119] "Regulatory elements" include promoters, enhancers, internal ribosome entry sites (IRES), and other expression control elements (e.g., transcription termination signals such as polyadenylation signals and polyU sequences). Such regulatory elements are described, for example, in Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif (1990), which is incorporated herein by reference in its entirety. Regulatory elements include those that induce direct constitutive expression of a nucleotide sequence in many types of source cells, and those that induce direct expression of a nucleotide sequence only in certain source cells (e.g., cell-specific regulatory sequences). Regulatory elements can also induce direct expression in a time-dependent manner, which may or may not be tissue- or cell-type-specific.

[0120] Protein expression or amount of the transcription factors described herein can be improved by contacting cells with an agent that activates or enhances expression of the transcription factor. In any embodiment, the agent is selected from the group consisting of nucleotide sequences, proteins, aptamers and small molecules, ribosomes, RNAi therapeutics and peptide nucleic acids (PNAs) and their analogs or variants. In certain aspects, the agent is exogenous. In some aspects, the agent is a small molecule. In some aspects, the nucleotide sequence is included as part of a transcription activation system (e.g., gRNA or TALEN for use in gene editing systems) to improve expression of one or more transcription factors. In some aspects, activation of the selected transcription factor can be performed in vivo using a transcription activation system (e.g., CRISPRa) to improve expression of one or more transcription factors.

[0121] Although the present invention has been described using human genes, other sources may be used as appropriate depending on the species. Codon optimization may also be performed to optimize expression, and expression vectors, mRNA or repRNA may also be optimized for use. Source cells may be targeted by using specific expression vectors and / or by selecting cell-specific promoters.

[0122] The present disclosure provides methods for converting source cells, such as Muller glia cells or astrocytes, by direct cell conversion or transdifferentiation into cone photoreceptor cells for use in treating retinal degenerations and diseases, including inherited retinal dystrophies such as age-related macular degeneration, retinitis pigmentosa, retinal damage, and retinopathies involving cone photoreceptor loss. Additionally, the present disclosure provides nucleic acid molecules and compositions for use in treating retinal degenerations or diseases.

[0123] Any of the methods described herein can have one or more, or all, steps performed in vitro, ex vivo or in vivo.

[0124] When the steps of the method described herein are carried out in vitro, the cell or cell population can be delivered in a pharmacy-acceptable ophthalmic formulation by intraocular injection.When administering the formulation by intravitreal injection, the solution can be concentrated, for example, so that a minimal volume can be delivered.The concentration for injection can be any amount that is effective and non-toxic, depending on the factors described herein.

[0125] The methods described herein may be utilized for autologous therapy (i.e., source cells are isolated from a subject, undergo transdifferentiation to produce cone photoreceptor cells, and then injected back into the same subject) or allogeneic therapy (i.e., source cells are isolated from a subject, undergo transdifferentiation to produce cone photoreceptor cells, and then injected back into a different subject).

[0126] Alternatively, the method described herein for source cell to cone photoreceptor cell is a method of in vivo cell transformation, whereby a nucleic acid molecule encoding at least one or more transcription factors is delivered to a subject, and the transformation or transdifferentiation of source cell to desired differentiated target cell occurs in vivo. Suitable methods for nucleic acid delivery for cell transformation for use with the present invention will include virtually any method by which nucleic acid (e.g., DNA or RNA) can be introduced into cells, as described herein or as would be known to those skilled in the art (e.g., Stadtfeld and Hochedlinger, Nature Methods 6(5):329-330 (2009); Yusa et al., Nat. Methods 6:363-369 (2009); Woltjen, et al., Nature 458, 766-770 (April 9, 2009)). Such methods include, but are not limited to, direct delivery of DNA by ex vivo transfection (Wilson et al., Science, 244:1344-1346, 1989; Nabel and Baltimore, Nature 326:711-713, 1987), optionally using lipid-based transfection reagents such as Fugene6 (Roche) or Lipofectamine (Invitrogen), by microinjection (Harland and Weintraub, J. Cell 2002, 111-112, 1987), or by cytotoxicity (Cell 2002, 111-112, 1988). Biol., 101:1094-1099, 1985; U.S. Patent No. 5,789,215, incorporated herein by reference), by injection (U.S. Patent Nos. 5,981,274; 5,945,100; 5,780,448; 5,736,524; 5,702,932; 5,656,610; 5,589,466 and 5,580,859, each of which is incorporated herein by reference); by electroporation (U.S. Patent No. 5,384,253, incorporated herein by reference; Tur-Kaspa et al., Mol. Cell Biol., 6:716-718, 1986; Potter et al., Proc. Nat'l Acad.Sci. USA, 81:7161-7165, 1984); by calcium phosphate precipitation (Graham and Van Der Eb, Virology, 52:456-467, 1973; Chen and Okayama, Mol. Cell Biol., 7(8):2745-2752, 1987; Rippe et al., Mol. Cell Biol., 10:689-695, 1990); by using DEAE-dextran followed by polyethylene glycol (Gopal, Mol. Cell Biol., 5:1188-1190, 1985); by direct sonication (Fechheimer et al., Proc. Nat'l Acad. Sci. USA, 84:8463-8467, 1987); liposome-mediated transfection (Nicolau and Sene, Biochim. Biophys. Acta, 721:185-190, 1982; Fraley et al., Proc. Nat'l Acad. Sci. USA, 76:3348-3352, 1979; Nicolau et al., Methods Enzymol., 149:157-176, 1987; Wong et al., Gene, 10:87-94, 1980; Kaneda et al., Science, 243:375-378, 1989; Kato et al., J. Biol. Chem., 266:3361-3364, 1991) and receptor-mediated transfection (Wu and Wu, Biochemistry, 27:887-892, 1988; Wu and Wu, J. Biol. Chem., 262:4429-4432, 1987); and any combination of such methods, each of which is incorporated herein by reference. The present invention is also directed to nucleic acid molecules, vectors, compositions and products that may be used with the disclosed methods. In one example, the composition comprises one or more nucleic acids encoding a transcription factor or variants thereof, and a pharma- ceutically acceptable carrier. The nucleic acid molecules, vectors, compositions and products according to the present invention may be introduced into the source cells by one or more expression vectors, for example, by lentivirus, adenovirus or AAV transduction. The nucleic acid molecules, vectors, compositions and products according to the present invention may be administered by intravitreal, suprachoroidal or subretinal injection. The expression vector may be administered by intravitreal, suprachoroidal or subretinal injection.

[0127] In a further example, the composition comprises a transcription factor or its variant and a pharma- ceutical acceptable carrier and is delivered as a protein therapeutic.Several methods have been developed to efficiently deliver proteins across the plasma membrane of photoreceptor or retinal pigment epithelium (RPE) cells in vivo, for example, using nucleolin present on the surface of photoreceptor cell bodies as a portal for delivering proteins to retinal cells upon intravitreal injection.

[0128] The pharma- ceutically acceptable carrier suitable for the delivery of the composition of the present invention and the method for its preparation are easily clear to those skilled in the art.Generally, the nature of the carrier will depend on the particular mode of administration used.For example, parenteral formulations usually contain injectable fluids, such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or vehicle, which contain pharma- ceutical and physiologically acceptable fluids.In addition to biologically neutral carriers, the pharmaceutical compositions to be administered can contain small amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives and pH buffering agents, such as sodium acetate or sorbitan monolaurate.

[0129] "Therapy", "treatment" and "treating" include both preventative and curative treatment of a condition, disease or disorder. It also includes slowing, preventing, controlling or stopping the progression of a condition, disease or disorder. It also includes preventing, curing, slowing, preventing, controlling or stopping the symptoms of a condition, disease or disorder. A "therapeutically effective amount" can vary depending on one or more of the subject and condition being treated, the weight and age of the subject, the severity of the condition, the method of administration, etc., which can be determined by one of skill in the art.

[0130] The terms "xeno-free (XF)" or "animal component-free (ACF)" or "animal-free", when used in relation to a medium, extracellular matrix, or culture condition, refer to a medium, extracellular matrix, or culture condition that is essentially free of xenogeneic animal-derived components. When culturing human cells, any proteins of a non-human animal, such as mouse, would be xenogeneic components. In certain embodiments, the xeno-free matrix may exclude mouse feeder cells or Matrigel™, and thus be essentially free of any non-human animal-derived components. Matrigel™ is a solubilized basement membrane preparation extracted from Engelbreth-Holm-Swam (EHS) mouse sarcoma, a tumor rich in extracellular matrix proteins, including laminin (major component), collagen IV, heparan sulfate proteoglycan, and entactin / nidogen.

[0131] The term "defined" when used in reference to a medium, extracellular matrix, or culture condition, refers to a medium, extracellular matrix, or culture condition in which the nature and amount of nearly all of the components are known. A "chemically defined medium" refers to a medium in which the chemical nature and amounts of nearly all of the components are known. These media are also called synthetic media.

[0132] A culture, matrix or medium is "essentially free" of certain reagents, such as signal transduction inhibitors, animal components or feeder cells, if the culture, matrix or medium, respectively, has levels of these reagents below detectable levels using conventional detection methods known to those of skill in the art, or if these agents are not added exogenously to the culture, matrix or medium.

[0133] Any medium, culture or matrix for any of the steps or sub-steps or throughout the process may be xeno-free or defined (as described herein). The medium may be chemically defined. One or more media or cultures for use according to the invention may be free or essentially free of any matrix components or may include a defined extracellular matrix or a xeno-free extracellular matrix. In the case of human cells, "xeno-free" means that the medium or culture is free or essentially free of any non-human animal components. Preferably, the ex vivo method of the invention is GMP compliant and uses xeno-free and / or defined cultures and media.

[0134] The indefinite articles "a" and "an," as used herein in the specification and claims, unless clearly indicated to the contrary, are to be understood to mean "at least one." Any ranges cited herein are inclusive.

[0135] As used herein in the specification and claims, in referring to a list of one or more elements, the phrase "at least one" or "at least one or more" should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed in the list of elements, nor excluding any combination of elements in the list of elements. This definition also allows for the optional presence of elements other than those specifically identified in the list of elements to which the phrase "at least one" refers, whether related or unrelated to the specifically identified element. Thus, as a non-limiting example, "at least one of A and B" (or, equivalently, "at least one of A or B" or, equivalently, "at least one of A and / or B") can refer in one embodiment to at least one, and optionally including more than one, A, where B is not present (and optionally including elements other than B); in another embodiment to at least one, and optionally including more than one, B, where A is not present (and optionally including elements other than A); in yet another embodiment to at least one, and optionally including more than one, A, and at least one, and optionally including more than one, B, (and optionally including other elements); etc.

[0136] Alternative descriptions of the present invention are described in the following numbered embodiments: 1. A method of converting a source cell into a cone photoreceptor cell by introducing at least one or more transcription factors or variants thereof selected from the group consisting of myocyte enhancer factor 2C (MEF2C), myocyte enhancer factor 2D (MEF2D), retinoid X receptor gamma (RXRG), and cone-rod homeobox (CRX) into the source cell, thereby converting the source cell into a cone photoreceptor cell.

[0137] 2. The method according to embodiment 1, wherein at least one or more transcription factors or variants thereof are encoded on one or more nucleic acids. 3. The method according to embodiment 1, wherein the at least one or more transcription factors or variants thereof are introduced into the source cell as transcription factor proteins.

[0138] 4. The method according to any one of embodiments 1-3, wherein the at least one or more transcription factors include at least two, or at least three, or at least four of MEF2C, MEF2D, RXRG and CRX, or variants thereof, or any combination thereof.

[0139] 5. The method according to any one of the preceding embodiments, wherein the at least one or more transcription factors are MEF2C, MEF2D, RXRG and CRX. 6. The method according to any one of the preceding embodiments, wherein the source cells are transdifferentiated into cone photoreceptor cells.

[0140] 7. The method according to any one of the preceding embodiments, wherein the source cells are Müller glial cells or astrocytes. 8. The method according to any one of the preceding embodiments, wherein the source cells are a population of source cells.

[0141] 9. The method according to any one of the preceding embodiments, wherein the source cell or population of source cells is derived from a mammal, preferably a human. 10. The method according to any one of the previous embodiments, wherein the at least one or more transcription factors are introduced into the source cell by lentivirus, adenovirus or AAV transduction.

[0142] 11. The method according to any one of the preceding embodiments, wherein the source cells are converted to cone photoreceptor cells if there is upregulation of any one or more cellular markers of cone photoreceptors, or downregulation of one or more cellular markers of the source cells, and / or one or more changes in cellular morphology from the source to the cone photoreceptors, or any combination thereof.

[0143] 12. The method according to embodiment 11, wherein the cell marker for cone photoreceptor cells is selected from one of cone arrestin (ARR3), GNAT2, and S / M / L opsin. 13. The method according to any one of the preceding embodiments, wherein at least 0.01%, at least 0.05%, at least 0.10%, at least 0.5% or at least 1.0% of the source cells are converted into photoreceptor cells.

[0144] 14. The method according to any one of the preceding embodiments, comprising culturing under suitable conditions for at least 4 days. 15. A cone photoreceptor cell produced by the method of any one of embodiments 1-14.

[0145] 16. A genetically modified cone photoreceptor cell produced by any one of embodiments 1-14, wherein the genetically modified cone photoreceptor cell is genetically modified with one or more exogenous nucleic acids encoding at least one or more transcription factors selected from the group including MEF2C, MEF2D, RXRG and CRX, or variants thereof.

[0146] 17. A nucleic acid molecule for converting a source cell into a cone photoreceptor cell, comprising a nucleic acid sequence encoding at least one or more transcription factors selected from the group consisting of MEF2C, MEF2D, RXRG and CRX or variants thereof.

[0147] 18. An exogenous expression cassette comprising one or more nucleic acid molecules according to embodiment 17, wherein the one or more nucleic acid molecules are operably linked to a promoter, preferably a cell-specific promoter.

[0148] 19. A cone photoreceptor cell according to embodiment 15 or 16, a nucleic acid molecule according to embodiment 17, or an exogenous expression cassette according to embodiment 18, for use in treating a retinal disease or retinal degeneration.

[0149] 20. A composition for converting a source cell into a cone photoreceptor cell, comprising at least one or more transcription factor proteins or variants thereof selected from the group consisting of MEF2C, MEF2D, RXRG and CRX, a cone photoreceptor cell according to embodiment 15 or 16, a nucleic acid molecule according to embodiment 17, or an exogenous expression cassette according to embodiment 18, and a pharma- ceutically acceptable carrier.

[0150] 21. The composition of embodiment 20 for use in treating a retinal disease or retinal degeneration. 22. A method of treating a retinal disease or retinal degeneration in a subject, comprising administering to the retina of a subject in need of treatment a therapeutically effective amount of at least one or more transcription factor proteins selected from the group consisting of MEF2C, MEF2D, RXRG and CRX or variants thereof, or a therapeutically effective amount of one or more nucleic acid molecules encoding at least one or more transcription factors, wherein an increase in at least one factor selected from the group consisting of MEF2D, MEF2C, RXRG and CRX or variants thereof induces conversion of a source cell into a cone photoreceptor cell, and the source cell is a Muller glia cell or an astrocyte.

[0151] 23. A method for treating a retinal disease or retinal degeneration in a subject, comprising administering a therapeutically effective amount of a cell or cell population according to embodiment 15 or 16 to the retina of a subject in need of treatment.

[0152] 24. A method for treating a retinal disease or retinal degeneration in a subject, comprising administering a therapeutically effective amount of the composition of embodiment 20 to the retina of a subject in need of treatment. 25. The method according to any one of the previous embodiments, wherein the retinal disease or degeneration is age-related macular degeneration (AMD), retinitis pigmentosa (RP) or diabetic retinopathy.

[0153] 26. A method according to any one of the preceding embodiments, wherein the method increases the number of cone photoreceptor cells in the retina. In an alternative definition, according to a first embodiment, the present invention provides a method for converting a source cell into a cone photoreceptor cell by introducing into the source cell at least one or more transcription factors or variants thereof selected from the group consisting of myocyte enhancer factor 2C (MEF2C), myocyte enhancer factor 2D (MEF2D), retinoid X receptor gamma (RXRG), and cone-rod homeobox (CRX), thereby converting the source cell into a cone photoreceptor cell.

[0154] In an alternative definition, according to a second embodiment of the present invention, there is provided a method of converting a source cell into a cone photoreceptor cell by introducing into the source cell at least one or more transcription factors or variants thereof selected from the group consisting of myocyte enhancer factor 2C (MEF2C), myocyte enhancer factor 2D (MEF2D) and retinoid X receptor gamma (RXRG); wherein the at least one or more transcription factors optionally further include cone-rod homeobox (CRX) or a variant thereof.

[0155] According to one embodiment, at least one or more transcription factors or variants thereof are encoded on one or more nucleic acids. According to an alternative embodiment, at least one or more transcription factors or variants thereof are introduced into the source cell as transcription factor proteins.

[0156] In an alternative definition, in some embodiments, the at least one or more transcription factors include at least two or at least three or at least four of MEF2C, MEF2D, RXRG and CRX, or variants thereof, or any combination thereof. In further embodiments, the at least one or more transcription factors include at least two or at least three of MEF2C, MEF2D and RXRG, or variants thereof, or any combination thereof. In another embodiment, the at least one or more transcription factors are MEF2C, MEF2D, RXRG and CRX.

[0157] In any embodiment, the source cells may be Müller glia cells and / or astrocytes. The source cells may be single cells or a population of source cells. The source cells or population of source cells may be from a mammal, preferably a human. The source cells may be transdifferentiated into cone photoreceptor cells according to any method of the present invention.

[0158] In a preferred embodiment, at least one or more transcription factors are introduced into the source cells by lentiviral, adenoviral or AAV transduction. In any of the methods of the invention described herein, the source cells are converted to cone photoreceptor cells if there is upregulation of any one or more cell markers of cone photoreceptors, or downregulation of one or more cell markers of source cells, and / or one or more changes in cell morphology from source to cone photoreceptors, or any combination thereof.Preferably, the cell markers of cone photoreceptors are selected from one of cone arrestin (ARR3), GNAT2, and S / M / L opsin.

[0159] In an alternative definition, the present invention also provides a cone photoreceptor cell produced by the method according to the present invention, or a genetically modified cone photoreceptor cell produced by the method according to the present invention, wherein the genetically modified cone photoreceptor cell is genetically modified with one or more exogenous nucleic acids encoding at least one or more transcription factors or variants thereof selected from the group including MEF2C, MEF2D, RXRG and CRX. Alternatively, the at least one or more transcription factors or variants thereof are selected from the group consisting of MEF2C, MEF2D and RXRG, and the one or more nucleic acids optionally further encode CRX or a variant thereof.

[0160] In an alternative definition, the present invention further provides a nucleic acid molecule for converting a source cell into a cone photoreceptor cell, comprising a nucleic acid sequence encoding at least one or more transcription factors or variants thereof selected from the group consisting of MEF2C, MEF2D, RXRG and CRX. Alternatively, the at least one or more transcription factors or variants thereof are selected from the group consisting of MEF2C, MEF2D and RXRG, and optionally further CRX or a variant thereof.

[0161] The present invention still further provides an exogenous expression cassette comprising one or more nucleic acid molecules according to the present invention, wherein the one or more nucleic acid molecules are operably linked to a promoter, preferably a cell-specific promoter.

[0162] In any of the methods described herein, the method can further comprise administering to an individual the cells, the nucleic acid molecule or the cell or cell population comprising the exogenous expression cassette produced according to the methods of the invention.

[0163] The present invention provides methods for use in the treatment of retinal diseases or degeneration, including the treatment of age-related macular degeneration (AMD), retinitis pigmentosa (RP) or diabetic retinopathy. In an alternative definition, the present invention also provides a composition for converting source cells into cone photoreceptor cells, comprising at least one or more transcription factor proteins or variants thereof selected from the group consisting of MEF2C, MEF2D, RXRG and CRX, and a pharmaceutically acceptable carrier.A further embodiment provides a composition for converting source cells into cone photoreceptor cells, comprising a cone photoreceptor cell or a population of cone photoreceptor cells produced according to the method of the present invention; a nucleic acid molecule produced according to the method of the present invention; or an exogenous expression cassette produced according to the method of the present invention; and a pharmaceutically acceptable carrier.The composition can be for use in treating retinal disease or retinal degeneration.

[0164] In an alternative definition, in any of the aspects of the methods of the invention described herein, the source cell is a Müller glial cell and the transcription factor or variant thereof is (a) MEF2C, MEF2D, RXRG and CRX; (b) MEF2C, MEF2D and RXRG; (c) MEF2C, MEF2D and CRX; (d) MEF2D, RXRG and CRX; (e) MEF2C, RXRG and CRX; (f) MEF2C and MEF2D; (g) MEF2D and RXRG; (h) MEF2D and CRX; (i) MEF2C and RXRG; (j) MEF2C and CRX; (k) RXRG and CRX; (l) MEF2D; (m) MEF2C; (n)RXRG; or (o)CRX It is.

[0165] The expression of transcription factor used herein is improved through cloning transcription factor gene into lentivirus and / or AAV expression vector.However, this is merely exemplary, and any expression vector can be used.Alternatively, mRNA or self-replicating / self-amplifying RNA (srRNA, saRNA or repRNA) can be used, and even intact functional protein can be used.

[0166] DNA, RNA and proteins can be introduced into cells in a variety of ways, including, for example, viral transduction, microinjection, electroporation and lipid-mediated transfection. Ophthalmic drug delivery is known to be a difficult challenge due to the presence of an effective epithelial barrier that protects ocular tissues. Viruses such as lentiviruses and AAVs are often injected into the vitreous or locally into the neural retina by subretinal injection to circumvent the epithelial barrier. RNA can be delivered to retinal cells using cell penetrating peptides (CCPs), which are synthetic and natural short amino acid sequences that can cross the cell membrane thanks to a transduction domain, e.g., TAT fusions, e.g., using the HIV-1-TAT protein. TAT has also been used for protein delivery. Other cell penetrating peptides (CPPs) are also known, and intact proteins can be delivered using CPPs as fusion proteins and even by non-covalent CPP / protein complexes.

[0167] At present, AAV is preferred for gene therapy because it is non-immunogenic, for example, LUXTURNA® (voretigene neparvovec-rzyl), which received marketing approval from the FDA in 2017 as an in vivo gene therapy for treating one subtype of retinitis pigmentosa, which involves AAV2 expressing RPE65. However, there is much research into non-viral delivery methods, such as plasmid DNA, lipid / nucleic acid lipoplexes, cationic liposomes, cationic polymers, nanoparticles and cationic metal complexes. Using genome editing techniques, such as CRISPR / Cas9, it is possible to selectively activate the required protein rather than cellular delivery of DNA, RNA or protein. CRISPR activation (CRISPRa) is an optimized method for overexpression of certain genes that uses the inactivated CRISPR-Cas9 system (dCas9) to upregulate target genes in their native context.

[0168] The above description is provided for illustrative purposes only and is not intended to limit the disclosure to the precise form disclosed. Details of one or more aspects of the disclosure are described above in the accompanying description. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the disclosure, the preferred methods and materials are described below. EXAMPLES

[0169] Example 1 – Identification of transcription factors involved in the transdifferentiation of Müller glia cells into photoreceptors Publicly available single-cell RNA sequencing data on adult human retina and retinal organoids (Cowan et al., Cell 2020) was added to the applicant's proprietary platform (see, e.g., WO2017106932). The platform predicted and ranked transcription factors involved in the transdifferentiation of macroglia, including Muller glia and astrocytes, into cone photoreceptors. Based on the percentage of network coverage and other criteria, the top four ranked transcription factors were identified as MEF2C, MEF2D, RXRG and CRX. These transcription factors were carried forward for in vitro, in vivo and ex vivo experiments.

[0170] Example 2 – Generation of lentiviruses encoding MEF2C, MEF2D, RXRG and CRX Lentiviral vectors were generated by cloning MEF2C, MEF2D, RXRG and CRX individually. Polycistronic vectors were also generated encoding combinations of MEF2C, MEF2D and RXRG (Figure 2) and combinations of MEF2C, MEF2D, RXRG and CRX. The transcription factors were under the control of the GFAP promoter and expressed GFP. The following expression cassettes were generated (in this case GFAPp stands for the GFAP promoter): GFAPp-MEF2C-GFP GFAPp-MEF2D-GFP GFAPp-RXRG-GFP GFAPp-CRX-GFP GFAPp-MEF2C-MEF2D-RXRG-GFP GFAPp-MEF2C-RXRGmyc GFAPp-MEF2D-CRX-mCherry Example 3 – Transduction of retinal organoid differentiation with transcription factors To assess whether MEF2C, MEF2D, RXRG and CRX, alone or in combination, could transdifferentiate Müller glial cells into cone photoreceptors, human retinal organoids were used as an in vitro model.

[0171] Human iPSCs were derived from fibroblasts using Yamanaka factors to generate stable iPSC lines (38F) and maintained in mTeSR medium. Retinal organoids were differentiated from iPSCs following the protocol of Cowan et al., Cell 2020. iPSC colonies were dissociated using EDTA (0.5 mM in PBS) at 37°C to generate embryoid bodies. Embryoid bodies were grown in suspension in 2 mL of mTeSR medium supplemented with 10 uM blebbistatin in petri dishes. Embryoid bodies were cultured for 125 days.

[0172] On day 125, retinal organoids were transferred to low attachment 96 well plates in 100ul of medium. Lentivirus mix was made up to 100ul in medium (RDM(N2)+FBS+taurine+0.5uM RA+1uM 9-cis RA+6ug / ml polybrene). Medium was removed from organoids and replaced by virus mix and returned to incubator. Retinal organoids were transfected with either GFAPp-GFP (control virus), GFAPp-MEF2C-GFP, GFAPp-MEF2D-GFP, GFAPp-RXRG-GFP, GFAPp-CRX-GFP, GFAPp-MEF2C-MEF2D-RXRG-GFP, GFAPp-MEF2C-RXRG or GFAPp-MEF2D-CRX-mCherry.

[0173] On the first day after transfection, the medium was brought to 200ul with (RDM(N2)+FBS+taurine++0.5uM RA+1uM 9-cis RA) and then at the end of the day the organoids were transferred to 25-well low attachment plates in 1ml of medium. From the second day after transfection onwards, the medium was changed twice a week for up to 4 weeks.

[0174] Example 4 – Colocalization of transcription factors with cone arrestins detected by immunochemistry On the 14th day after transfection, the retinal organoids were washed, cryopreserved, and then sectioned onto slides. Immunofluorescence staining was performed on 10 μm thick frozen sections by using a primary antibody against ARR3, a cone-specific marker. The slides were then incubated with a secondary antibody containing a fluorophore to visualize cells expressing ARR3.

[0175] Fluorescent imaging of the slides was performed to analyze the expression of GFP and ARR3 under a Leica epifluorescence microscope and a Zeiss LSM 910 confocal microscope. Successful reprogramming events were determined by colocalization of GFP, driven by the GFAP promoter and linked to transcription factor expression, with a cone photoreceptor specific marker (ARR3), which was not observed in controls, but in organoids receiving the GFAP_TF_GFP vector, clear colocalization between GFP and ARR3 was observed, indicating that conversion had occurred (GFP+ / ARR3+).

[0176] No expression of GFP was observed in cone photoreceptors following transduction of human retinal organoids with a control lentivirus expressing GFP, see Figure 1 (A and B). Co-expression of GFP and ARR3 was observed following transduction of human retinal organoids with lentiviral vectors encoding MEF2C (Figure 1 (C and D)), MEF2D (Figure 1 (E and F)), RXRG (Figure 1 (G and H)) alone, and the combination MEF2C-MEF2D-RXRG (Figure 3). Importantly, the morphology of the transformed cells was consistent with photoreceptors of the outer nuclear layer, although in this case primitive outer segments were also observed. No evidence of co-expression of GFP and ARR3 was observed following transduction of human retinal organoids with the corresponding lentiviral vector encoding CRX.

[0177] Quantification of total cone receptors (ARR3+), total GFP+ cells, and reprogrammed cones (ARR3+ / GFP+) in human retinal organoids after transduction of the three transcription factors is shown in Figure 4. It should be noted that human retinal organoids also contain intact, pre-existing cones (ARR3+) and that there is some inherent variability between retinal organoids.

[0178] Example 5 –Transduction of mouse retinal explants with transcription factors To further evaluate transcription factor transduction in vivo, reprogramming of Müller glia into cone photoreceptors was performed in postmortem mouse retinal explants.

[0179] Figure 5 (A and B) shows that when a control AAV driving mCherry expression under the GFAP promoter was used, no successful reprogramming events of Müller glia to cone photoreceptors were observed (white arrowheads exemplify areas where mCherry-marked photoreceptors would be expected to be found). Alternatively, when an AAV2 variant encoding two transcription factors (MEF2D_CRX) predicted by the MOGRIFY v.2.5 system was used (C and D), colocalization of mCherry with ARR3 was observed, exemplifying the transcription factor driving the conversion of Müller glia to cone (arrows exemplify mCherry-marked photoreceptors, where photoreceptor morphology is clearly visible).

[0180] Figure 6 shows quantification of total cone photoreceptors (ARR3+), total GFP+ cells, and reprogrammed cones (ARR3+ / GFP+) in mouse retinal explants after transduction with AAVs encoding factors predicted by the MOGRIFY v.2.5 system. No successful reprogramming of Müller glia to cones was observed when a control vector was used, however, successful reprogramming of Müller glia to cones (ARR3+ / mCherr+) was observed when a bicistronic AAV2 variant vector encoding two transcription factors (MEF2C_RXRG and MEF2D_CRX) was used.

[0181] Example 6 – Light sensitivity and electrical output of ex vivo human retinal explants AAVs encoding transcription factors were also evaluated in cadaveric human retinal explants by measuring their light sensitivity and electrical output before and after viral transduction. In Figures 7A, 7B, 8A, and 8B, the increased light sensitivity of human retinal explants after treatment suggests promising therapeutic value.

[0182] The explants were placed ganglion cell layer down onto the central recording electrode grid of a multi-electrode array (60MEA200 / 30iR-TI, Multi Channel Systems, GmbH: TiN electrodes, SiN insulating layer, TiN contact pads and tracks. Electrodes 30 μm diameter, 200 μm spacing, "square packing" with internal reference electrode). After the explant was centered on the array grid, a "harp" (a ring of pure platinum, supporting a nylon mesh) was placed over the explant to prevent it from floating away when medium was added. 800ul of complete Neurobasal-A (47ml Neurobasal A, 1ml 50x B27 supplement, 500ul 100x N2 supplement, 500ul 100mM sodium pyruvate, 500ul Glutamax, 50ul 50nM taurine, 60.8ul 1mM N-acetyl-L-cysteine ​​and 500ul 100x Pen / Strep) containing 1um 9 cis-retinal was added to the MEA wells. The arrays were incubated in a 37°C incubator (5% CO 2 ) in tissue culture dishes. The medium was changed every 2 days. Electrophysiological recordings are performed before transduction, usually the day after detachment, and 14 days after transduction. The MEAs were placed into a twin MEA "clamshell" amplifier (MEA2100-2x60-System). Once placed in the recording device, the explants on the array were further sealed from the immediate surroundings with the aid of mini bioincubators (Ndimension (Science and Engineering) Ltd.) that have been 3D printed using biocompatible polymers. Two types of mini bioincubators are used: opaque / matt black for "dark-adapted" recordings and transparent / "colorless clear" for "light-adapted recordings". These devices are maintained in a 5% CO2 in sterile air (110 cm) atmosphere to maintain the pH of the bicarbonate-buffered culture medium. 3The MEA was equipped with a port for the delivery of 1000 Hz (100 / min). A solid-state sensor module (ExplorIR-WH-20 Gas Sensing Solutions) was incorporated to acquire data on temperature, relative humidity and CO2 concentration (in %) or ppm of the air above and surrounding the MEA at a sampling rate of 2 Hz. The temperature of the fluid in the MEA chamber was kept at 37°C (±0.1°C) by a heating plate integrated into a TC02 temperature controller unit (Multi Channel Systems) and an MEA2100-2x60 amplifier. Wideband neural (1Hz-3.3KHz) signals were acquired from the MEA using a data acquisition system centered on a freely programmable Digital Signal Processor Interface (MCS-IFB; ADC resolution: 16 bits; USB 3.0 link: Multi Channel Systems). Data were acquired at a sampling rate of 25KHz per channel using the MCRack package (version: 4.6.2.Multi Channel Systems) and stored on disk. Two sets of second-order IIR Butterworth filters were provided in the MCRack virtual instrument for display both during recording and subsequent "offline" analysis. The first filter, with a bandpass of 300Hz-3.3KHz, isolates the spiking activity of the retinal ganglion cells (RGCs), and the second filter, with a bandpass of 1-20Hz, reveals the mERG waveform. A "bandstop" or "notch" filter with f=50Hz is also used to remove any "hum" (Q=2.0). Thus, two "data stream objects" are generated for the "offline" analysis, one for the spiking activity of the RGCs and one for the mERG slow waves. The specimens were conditioned and dark-adapted for 1 hour before recording began. Exposures were performed using a dedicated LED-based "light stimulator", with light delivered via twin fiber-optic light pipes to illuminate the underside of the explant. Stimulation was "full-field" (i.e., the entire specimen).The intensity, pulse width and frequency of the light flashes were controlled by external electronics, while TTL pulses marking the time of stimulation were sent to the data acquisition system from a special interface box. The light exposures were always performed in the same sequence: 15 ms red flash, 15 ms green flash, 15 ms blue flash, 15 ms white flash, 1 s white flash. The flashes were spaced 30 s apart. Responses to three flashes at each of 15 increasing light intensity steps were recorded at each intensity (for later "averaging" purposes). These intensity steps were designed to cover the full range of both "dark-adapted" and "light-adapted" vision. Five minute light-free intervals were used between the tones to allow the explants to "recover" especially from any "photobleaching" effects. Once all recordings under dark adaptation were completed, the room lights were switched on to allow the explants to light adapt, and the opaque / matt black mini bioincubator was replaced with a transparent / "colorless transparent" equivalent to allow ambient light to enter. This light adaptation period lasts 15-30 min. All tones and white recordings are repeated. The RGC spike activity data stream objects were analyzed using the NeuroExplorer package (V.5.129.64-bit version Nex Technologies). To translate unit activity compatible with import into the NeuroExplorer package, which is designed to consider action potential firing as a "point process", it is necessary to extract the "detected spikes" and represent them as "timestamps". This is done by directing the filtered spike data stream through MCRack's "Spike Sorter" virtual instrument. Within this instrument, a detection voltage threshold was set with a polarity that matched the dominant polarity represented by the spike waveform; this threshold was set to four standard deviation units about the RMS noise level estimated by the software.The second element of the Spike Sorter instrument generates a "spike cutout" of the detected spike waveform, usually with 1 ms pre-trigger (threshold crossing), 2 ms post-trigger of the digitized signal, and a 2 ms "dead time" within which a second spike cannot be registered. NeuroExplorer's Default Timestamp Frequency of 20,000 Hz is changed to match the MCRack digitization frequency of 25 KHz per channel. Spike timestamps and logs of TTL pulses from the light stimulator control interface are processed within NeuroExplorer to generate both continuous running time histograms and pre- and post-stimulus time histograms of this activity for all electrodes in parallel (1 second bins), and this information is then ported to Excel for subsequent graphing and comparative analysis. Data generated from any individual electrode channel is logged at this point, displaying correlates of the degenerative process that "marshes" the recording. The mERG data stream objects were analyzed in two stages. First, the frequency of "thresholded" mERGs was determined in a manner equivalent to the previous detection of RGC spikes, where data was directly ported to Excel via a text file intermediate for subsequent graphing and analysis. In contrast, information regarding the relative amplitude and occurrence time of the mERG "a" and "b" wave components was determined using a combination of "peak / trough" detection and "active cursors" in MCRack and Spike 2 (Cambridge Electronic Design V8.07x64). The varying mERG profiles for both "control" and "treated" retinal explants under different light stimulation conditions (light / dark adaptation, tone, intensity) and over time of transduction were examined, and the relative "a" and "b" wave fingerprints were determined via scatter plot and cluster analysis.

[0183] Figures 7A and 7B show human retinal explants tested for light responsiveness using a multi-electrode array (MEA) before transduction and 2 weeks after transduction with either AAV_GFAP_mcherry (control, A) or AAV_GFAP_MEF2C_RXRG and AAV_GFAP_MEF2D_CRX (TF-treated, B). In Figure 7A, mERG Awsum decreased over the 2-week period of culture after transduction in comparison to baseline (P=0.088, NS, paired t-test). In Figure 7B, mERG Awsum increased significantly over the 2-week period after transduction with TF (p≦0.0001, paired t-test). These findings suggest that there was an increase in light-responsive cells (i.e., photoreceptors) over the 2-week period following transduction with TF, but not the mCherry control. Recordings were made under light-adapted conditions using colored light, thus excluding the light responses of rod photoreceptors and further selecting for the responses of cone photoreceptors.

[0184] Figure 8 quantifies the number of MEA channels registering mERG. There were 60 channels on the MEA chip, but one was a reference electrode, so the maximum number of channels that can be recorded is 59. In Figure 8A, when explants were treated with GFAP_mCherry control AAV, the number of channels recording mERG responses dropped over the 2-week culture period after transduction, whereas in Figure 8B, when explants were treated with GFAP_MEF2C_RXRG AAV and GFAP_MEF2D_CRX AAV, there is an increase in the number of channels recording mERG. These findings indicate that the number of light responses was increased across the MEA, and that mERG is detected after 2 weeks of treatment in channels that did not record mERG at baseline. This is consistent with the generation of new cones by transforming cells due to delivery of MEF2C, MEF2D, RXRG, and CRX.

[0185] Example 7 - ARR3+ cells can be observed outside the external nuclear layer following in vivo delivery of transcription factors Reprogramming of Müller glia into cone photoreceptors by in vivo delivery of transcription factors was evaluated in C57BL / 6JRj male mice to validate the findings of successful conversion of Müller glia into cones observed in mouse retinal explants described in Example 5. All mice were transfected with 2×10 6 AAVs encoding β-catenin (GFAP_βcatenin_GFP) for activation and induction of GFAP expression in Müller glial cells prior to delivery of TFs. 9 Treatment was on day 0 with intravitreal (IVT) administration of 1 vector genome copy / eye.

[0186] For intravitreal injections, anesthetized animals were placed under a stereoscope and a small intrascleral incision was made near the limbus using a 30G needle to expose the vitreous cavity. A total volume of 1 ul of AAV vector was injected into the intravitreous space using a microsyringe fitted with a 33G needle.

[0187] On day 14, the mice received a second injection. At this point, the mice receiving the second injection were divided into three groups. The first group received 2 × 10 9 The second group received an IVT injection of two bicistronic TF containing AAV2 variant vectors (GFAP_MEF2C-RXRG_myc and GFAP_MEF2D_CRX_mCherry) at a total dose of 2 × 10 vector genome copies / eye. 9 The third group received subretinal injections of two bicistronic TF containing AAV2 variant vectors (GFAP_MEF2C-RXRG_myc and GFAP_MEF2D_CRX_mCherry) at a total dose of 2 × 10 vector genome copies / eye. 9 received the AAV2 variant control AAV(GFAP_mCherry), encoding the mCherry fluorophore, at a total dose of 1 vector genome copies / eye.

[0188] For subretinal injections, anesthetized animals were placed under a stereoscope and a small incision with a 30G needle was made on the temporal side of the conjunctiva / sclera to expose the choroid. Using the same needle, a small opening was made on the temporal side of the choroid. The cornea was punctured to reduce intraocular pressure. A Hamilton microsyringe was filled with 1 μl solution and introduced through the exposed choroid into the subretinal space. The solution was injected into the subretinal space and injection was confirmed using in vivo SD-OCT imaging.

[0189] At the termination time point of day 42, animals were sacrificed and eyes were enucleated. Eyes were immersion fixed in 4% paraformaldehyde and the posterior eye cups were cryoprotected in an ascending gradient of sucrose. Following this, eye cups were embedded in optimal cutting temperature compound (OCT) and frozen in liquid nitrogen-cooled isopentane for histological and immunohistochemical examination. Immunofluorescence staining was performed on 12 μm-thick cryosections using primary antibodies against the cone-specific markers ARR3 and mCherry for visualization of GFAP_MEF2D_CRX_mCherry. Slides were then incubated with fluorescent secondary antibodies and examined for ARR3 and mCherry expression / localization using a Zeiss LSM 910 confocal microscope. ARR3+ cells outside the photoreceptor layer were quantified by manual counting during microscopy.

[0190] Immunohistochemical detection of ARR3 and mCherry in mouse retinal sections from these three groups of AAV-treated mice, as well as from "no-injection wild-type control mice", showed that ARR3+ cells could be identified outside the photoreceptor layer in all four groups. A very low baseline of ARR3+ cells was observed in a small number of retinal sections from the AVV control group and the "no-injection wild-type control" group. However, we observed a trend toward increased numbers of ARR3+ cells outside the outer nuclear layer (ONL) with TF treatment, regardless of the route of administration (subretinal vs. intravitreal).

[0191] The somas / nuclei of these ARR3+ cells were located in the INL, not the ONL, as expected for cone photoreceptors. However, the location of the INL is consistent with that of Müller glial nuclei, thus suggesting that they may represent transformed Müller glial cells. Together, this suggests that new cone photoreceptors can be formed following TF delivery to the eye. Overall, this is consistent with the Müller glia reprogramming events identified in transduced retinal organoids and mouse retinal explants.

[0192] In contrast to observations from TF-treated retinal organoids and mouse retinal explants, colocalization of mCherry with ARR3 was not observed in either TF-treated or AAV-control groups. These differences may be due to variability in the time points examined (14 days post-transduction for retinal organoids and mouse retinal explants). Earlier time points may have captured converting "Müller glia-to-cone" hybrid cells that still had significant GFAP promoter activity. On the other hand, time points later than "4 weeks post-TF delivery" in vivo may only capture cells that are much more cone-like, in a situation where the GFAP promoter is no longer active, resulting in the cessation of expression of the mCherry fluorophore. The lack of mCherry expression in ARR3+ cells outside the photoreceptor layer may be because conversion was independent of the GFAP_MEF2D_CRX_mCherry AAV and mediated exclusively by the GFAP_MEF2C-RXRG_myc AAV (not detectable by immunohistochemistry).

[0193] No mCherry signal was observed in photoreceptors within the photoreceptor layer in any of the groups examined, supporting Müller glia-specific expression of the promoter system used.

[0194] Example 8 – Contribution of transcription factors analyzed in transcriptome analysis of 2D Müller glia Steps for deriving Müller glial cells: On day 125 of retinal organoid differentiation, individual organoids were dissociated into single cell suspensions using the Neurosphere Dissociation Kit (Miltenyi Biotec - 130-095-943) according to the manufacturer's protocol. The Müller glia population was cultured at 2ug / cm 2 Retinal organoids were grown by seeding single-cell suspensions onto bovine fibronectin-coated T25 flasks at a concentration of 100 mM NaCl. Adherent cells were grown at 37°C and 5% CO in medium composed of DMEM high glucose, 10% fetal bovine serum, human EGF 40 ng / ml, and human bFGF 40 ng / ml. 2 Müller glia were grown in 2D MG medium (hereafter referred to as 2D MG medium) for approximately 2-3 weeks until 90% confluence of T175 was achieved. To cryopreserve confluent T175 cultures, Müller glia were dissociated using TrypLE and cryopreserved in aliquots of 1x10^6 cells in DMEM high glucose, 10% fetal bovine serum, and 10% dimethyl sulfoxide.

[0195] To evaluate the composition of retinal organoid-derived cultures, RT-qPCR was performed for markers of various retinal cell types.Following cryopreservation, total RNA was extracted from vials of frozen cells using Arcturus PicoPure RNA isolation kit, and cDNA was generated from the extracted RNA using Bioline SensiFAST™ cDNA synthesis kit.qPCR was performed on the resulting cDNA to confirm the expression of Müller glia markers CRYM, GLUL, VIM and PAX6, and the absence of photoreceptor markers ARR3, GUCA1C and NR2E3.

[0196] Generation of RNA samples: Retinal organoid-derived Müller glial cells were seeded into 6-well plates at a density of 10,000 cells / well and maintained in 2D MG medium. 24 hours after seeding, cells were transduced in quadruplicate with lentiviral vectors encoding one of the following five expression cassettes: ·GFAP_MEF2C_MEF2D_RXRG_GFP EF1a_GFP EF1a_CRX_GFP EF1a_RXRG_GFP EF1a_MEF2D_GFP EF1a_MEF2C_GFP To promote transduction, each viral vector was combined with 2 ml of 2D MG medium supplemented with 6 ug / ml polybrene and incubated at 37°C and 5% CO. 2 The cells were incubated at RT overnight at RT. Transduction efficiency was >90% based on GFP expression. After 24 h, viral supernatant was aspirated and replaced with fresh 2D MG medium. Seven days after transduction, total RNA was extracted from the cells using the Arcturus PicoPure RNA isolation kit according to the manufacturer's protocol.

[0197] RNA-Seq libraries were prepared and sequenced at a depth of 12G per sample on an Illumina NovoSeq 6000 from Novogene (Cambridge). Reads were demultiplexed and aligned to the human genome (GRCh38) via STAR (version 2.7.10b), and transcript counts were performed via RSEM (version 1.3.3). Differential gene expression analysis was performed via DESeq2 (version 1.26.0). Genes were considered significantly differentially expressed between TF-treated conditions and controls if they had a p-value of 0.05 or less and an absolute log fold change of >1.

[0198] Analysis of RNA samples: We observed statistically significant up- and down-regulation of genes in response to the addition of MEF2C, CRX or polycistronic (MEF2C, MEF2D, RXRG), but not when MEF2D or RXRG was added alone (Figure 12).Surprisingly, MEF2C alone and polycistronic (MEF2C, MEF2D and RXRG) each resulted in a much more significant up- and down-regulated number of genes than CRX alone.The number of differentially expressed genes increased between MEF2C alone and polycistronic, suggesting the synergistic effect of MEF2C, MEF2D and RXRG.

[0199] When we investigate the expression of genes that are statistically significantly upregulated in the reference scRNAseq retinal organoid data set of Cowan et al., we find that the upregulated genes of polycistronic, MEF2C alone and CRX alone are much more highly expressed in reference RPE (i.e. pigmented) cells than any other reference cell type (Figure 13). In MEF2C treatment conditions, a subset of the upregulated differentially expressed genes is also upregulated in reference proliferating cells. In polycistronic treatment conditions, we also observe that a subset of the upregulated differentially expressed genes is also upregulated in reference cone photoreceptors. Figure 13 also highlights how MEF2C and polycistronic (MEF2C, MEF2D and RXRG) each upregulate far more genes in the reference scRNAseq retinal organoid data set of Cowan et al. than CRX.

[0200] The effect of polycistronic, MEF2C alone and CRX alone on upregulating genes related to RPE cells raises the question of whether any RPE functions could be activated by these transcription factors. Retinal pigment epithelial (RPE) cells have several well-defined functions, including phagocytosis of cone photoreceptor outer segments; visual cycle (the biochemical process that regenerates chromophores); and pigmentation. Genes involved in phagocytosis (binding) by RPE cells include ITGAV, ITGB5, MFGE8 and CD81. Genes involved in phagocytosis (internalization) by RPE cells include MERTK, GAS6 and PTK2. Genes involved in the visual cycle of RPE cells include RLBP1, RPE65, LRAT, RDH5, RDH10 and RDH11. Genes involved in pigmentation of RPE cells include PMEL, TYR, TYRP1, GPR143, DCT, OCA2, RAB38 and MYRIP.

[0201] Figure 14 shows the effect of each transcription factor condition on genes involved in RPE function based on bulk sequencing data from 2D MG cultures. This analysis suggests that genes related to phagocytosis (binding) may be upregulated by MEF2C and polycistronic (MEF2C, MEF2D, and RXRG) to a greater extent than by CRX alone (which appears to have some effect), and apparently to a greater extent than MEF2D and RXRG. CRX alone may have the greatest effect in this system on upregulating genes related to the visual cycle and / or pigmentation, raising the possibility that an additive effect of CRX when combined with MEF2C or polycistronic may upregulate genes involved in diverse RPE functions. However, because these data are generated from bulk sequencing experiments, their interpretation is complicated by the possibility of divergent effects on MG. For example, if any transcription factor directs cells to transdifferentiate into more than one cell type (e.g., via pluripotent, proliferative and / or progenitor cells), genes underlying key RPE functions may be upregulated in some cells that adopt an RPE-like phenotype, while downregulated in other cells that adopt another cell phenotype (such as a cone-like phenotype).Thus, these data suggest that some RPE functions may be differentially activated by MEF2C, polycistronics and CRX, but require further investigation in systems where RPE-like cells can be isolated and specifically analyzed.

[0202] Example 9 – Transcriptomics of retinal organoids Retinal organoids were generated from the 38F iPSC cell line as described (Cowan et al., 2020). 16-week-old retinal organoids were treated with 1 mm purmorphamine to activate Müller glia. Retinal differentiation medium (RDM supplemented with FBS, N2, and taurine) and fresh purmorphamine were replenished every 2–3 days for a total of 2 weeks.

[0203] Individual organoids were transduced in 96-well plates containing RDM containing 6 mg / ml polybrene and supplemented with 0.5 mm 9-cis-retinoic acid and 2.5E+06 IVP lentivirus. A total of 16 retinal organoids were transduced with polycistronic v551 lentivirus (encoding MEF2C, MEF2D, and RXRG) and 6 with control GFP (v770) lentivirus.

[0204] The transduced organoids were maintained for 3 weeks with medium changes every 2–3 days. The transduced organoids were dissociated (Neurosphere Dissociation Kit, Miltenyi Biotech) and fluorescent positive cells were isolated by FACS sorting (BD FacsAria III).

[0205] FACS sorted cells were mixed with spike-in cells, iPSCs and k562 and loaded into a 10X Genomics Chromium instrument according to the manufacturer's instructions (10X Genomics, chemistry version 3.1). TF-treated and control samples were processed in parallel lanes. Single-cell RNA-Seq libraries were prepared according to the manufacturer's instructions (10X Genomics, chemistry version 3.1) and sequenced at a depth of at least 50,000 reads per cell on an Illumina NovoSeq 6000 from Novogene (Cambridge). Reads were demultiplexed and aligned to the human genome (GRCh38) and empty barcodes were removed via Cell Ranger (version 6.0.0). Low quality cells were removed and counts were normalized as per usual practice for scRNAseq analysis, see e.g. Basics of Single-Cell Analysis with Bioconductor, Robert Amezquita et al., version: 1.6.2 (chapters 1-3). Batch effects were assessed using spike-in cells that were demonstrated to have no batch effect. At this point, spike-in cells were removed from the dataset and normalized counts were recalculated. The remaining data were clustered using a shared nearest neighbor based unsupervised clustering method. Each cluster was annotated with cell type based on expression of unambiguously described retinal cell type markers.

[0206] In TF-treated conditions, we observed the presence of an RPE-like population that was absent in control (GFP-treated) conditions (see FIG. 13). As shown in FIG. 14, this RPE-like group upregulated canonical RPE markers, including bestrophin 1 (BEST1), melanocyte-inducing transcription factor (MITF), and pre-melanosome protein (PMEL).

[0207] Because the RPE-like cells in this example were identified using single-cell sequencing, the expression of genes related to RPE function can be analyzed at single-cell resolution in the RPE-like population without the complicating effects of other cell types that may be represented in the bulk sequencing data of Example 8. Thus, the expression of genes underlying the same well-defined functions, including phagocytosis of cone photoreceptor outer segments; visual cycle (the biochemical process that regenerates chromophores); and pigmentation, was analyzed in the RPE-like population of cells generated by TF treatment of retinal organoids. We observed upregulation of the expression of genes involved in each of these RPE functions in transcription factor (TF)-treated RPE-like populations versus controls (Figure 16). We observed the most clear improvement in pigmentation-related genes. These data suggest that RPE-like cells generated by polycistronic (MEF2C, MEF2D and RXRG) can exhibit RPE functional properties related to phagocytosis, visual cycle and pigmentation. Furthermore, since the data in Example 8 suggest that a significant proportion of the effects of the polycistronic (MEF2C, MEF2D and RXRG) complex are exhibited following treatment with MEF2C alone, it is believed that MEF2C alone may be sufficient to generate RPE-like cells exhibiting RPE functional characteristics associated with phagocytosis, the visual cycle and pigmentation.

[0208] Example 10 – Enhanced brain-derived neurotrophic factor secretion from TF-treated retinal organoid-derived Müller glial cells Transcriptomic data of TF-treated cells and retinal organoids described in Examples 8 and 9 show that addition of lentiviral-delivered GFAP_MEF2C_MEF2D_RXRG_GFP induces transdifferentiation of a subset of Müller glia into cells with an RPE-like transcriptional profile.

[0209] The "golden standard" for characterizing RPE cells is usually based on the fact that RPE cells are specialized epithelial cells that polarize and differentiate when located in a monolayer, resulting in significant transepithelial resistance. In addition, evaluation of specialized functions such as melanosome production and the presence of RPE markers, such as bestrophin 1 and RPE65, that are only found in fully differentiated epithelialized RPE cells, are usually also part of the evaluation. However, these classical RPE functions / markers are not all meaningful to explore in the present setting, because we are converting individual Müller glia cells into single individual RPE-like cells, which are not embedded in a monolayer and therefore are not expected to be able to polarize and differentiate to such a level within the timeline of the experiment to be performed.

[0210] Another well-established function of RPE is the secretion of several protein factors, including brain-derived neurotrophic factor (BDNF), so functional evaluation of Müller glia-derived RPE-like cells through the evaluation of the secretion levels of established RPE secretory factors is more meaningful.

[0211] Enhanced BDNF expression is seen in retinal organoid-derived Müller glial cells treated with polycistronic or monocistronic MEF2C vectors compared to other individual TFs and "no transduction" and transduction controls. To assess BDNF secretion levels from TF-treated Müller glial cells, BDNF levels were quantified in conditioned culture medium by enzyme-linked immunosorbent assay (ELISA).

[0212] Retinal organoid-derived Müller glial cells were derived as in Example 8 and plated in 12-well plates at 10,000 cells / cm. 2Cells were seeded at a density of 1000 x 1000 and maintained in 2D MG medium (described in Example 8). 24 hours after seeding, cells were transduced in triplicate with lentiviral vectors encoding either GFAP_GFP control or GFAP_MEF2C_MEF2D_RXRG_GFP. To facilitate transduction, each viral vector was combined with 1 ml of 2D MG medium supplemented with 6ug / ml polybrene and incubated at 37°C and 5% CO 2 The cells were incubated overnight at 100°C for 1 hour. The next day, the viral supernatant was aspirated and replaced with 1 ml of fresh 2D MG medium. Four days after transduction, the medium was replenished with an equivalent 1 ml volume of 2D MG medium. On day 6, the spent medium was aspirated and replaced with 500 ul of 2D MG medium, and then the supernatant was harvested on day 9. The supernatant for quantification of BDNF secretion was collected, centrifuged at 2000g for 10 minutes at 4°C, diluted 1:2, and then analyzed using the Human BDNF SimpleStep ELISA® kit.

[0213] BDNF secretion from Müller glia treated with a polycistronic lentiviral vector encoding the three TFs (GFAP_MEF2C_MEF2D_RXRG_GFP) was significantly increased (4-fold, p=0.0002) compared to cells treated with the GFP-only transduced control (GFAP_GFP) (Figure 18), suggesting that TF-treated Müller glia acquire RPE functions.

Claims

1. A nucleic acid molecule comprising a promoter operably linked to a nucleic acid sequence encoding myocyte enhancer factor 2C (MEF2C) or a functional variant thereof, wherein the promoter is for expression of MEF2C in macroglia.

2. The nucleic acid molecule of claim 1, wherein the functional variant of MEF2C comprises an amino acid sequence that is at least 90% identical to SEQ ID NO:

1.

3. A nucleotide sequence encoding MEF2C or a functional variant thereof, (a) SEQ ID NO:3, (b) SEQ ID NO:2, or (C) a nucleotide sequence having at least 60% identity to SEQ ID NO:3 or SEQ ID NO:

2. The nucleic acid molecule of claim 1 or 2, comprising:

4. 4. The nucleic acid molecule of any of claims 1 to 3, comprising a promoter operably linked to a nucleic acid sequence encoding one or more transcription factors selected from the group consisting of myocyte enhancer factor 2D (MEF2D), retinoid X receptor gamma (RXRG) and cone-rod homeobox (CRX), or functional variants thereof, or a combination thereof, wherein the promoter is for expression of the one or more transcription factors in macroglia.

5. 5. The nucleic acid molecule of claim 4, wherein a nucleic acid sequence encoding one or more transcription factors selected from the group consisting of myocyte enhancer factor 2D (MEF2D), retinoid X receptor gamma (RXRG) and cone-rod homeobox (CRX), or a functional variant thereof, or a combination thereof, is operably linked to the same promoter as a nucleic acid sequence encoding myocyte enhancer factor 2C (MEF2C) or a functional variant thereof.

6. (a) the functional variant of MEF2D comprises an amino acid sequence at least 90% identical to SEQ ID NO:4; and / or (b) the functional variant of RXRG comprises an amino acid sequence at least 90% identical to SEQ ID NO:7; and / or (c) the functional variant of CRX comprises an amino acid sequence at least 90% identical to SEQ ID NO: 10; A nucleic acid molecule according to claim 4 or 5.

7. A nucleotide sequence encoding MEF2D or a functional variant thereof, (a) SEQ ID NO:6, (b) SEQ ID NO:5, or (c) a nucleotide sequence having at least 60% identity to SEQ ID NO:6 or SEQ ID NO:

5.

7. The nucleic acid molecule according to claim 4, comprising:

8. The nucleotide sequence encoding RXRG or a functional variant thereof is (a) SEQ ID NO:9, (b) SEQ ID NO:8, or (c) a nucleotide sequence having at least 60% identity to SEQ ID NO:9 or SEQ ID NO:

8.

8. The nucleic acid molecule according to any one of claims 4 to 7, comprising:

9. A nucleotide sequence encoding CRX or a functional variant thereof, (a) SEQ ID NO: 12, (b) SEQ ID NO: 11, or (c) a nucleotide sequence having at least 60% identity to SEQ ID NO: 12 or SEQ ID NO:

11.

9. The nucleic acid molecule according to any one of claims 4 to 8, comprising:

10. 10. A nucleic acid molecule according to any one of claims 4 to 9, comprising a nucleotide sequence encoding MEF2C and a nucleotide sequence encoding RXRG.

11. 11. The nucleic acid molecule of claim 4, comprising a nucleotide sequence encoding MEF2C, a nucleotide sequence encoding MEF2D, and a nucleotide sequence encoding RXRG.

12. 12. A nucleic acid molecule according to any one of claims 1 to 11, wherein the nucleic acid sequence encoding MEF2C is 5' to a nucleic acid sequence encoding one or more further transcription factors.

13. 13. The nucleic acid molecule of claim 4, wherein the nucleic acid sequence encoding RXRG is 3' to the nucleic acid sequence encoding MEF2C.

14. 14. A nucleic acid molecule according to any one of claims 4 to 13, wherein a nucleic acid sequence encoding MEF2C is 5' to a nucleic acid sequence encoding MEF2D, and a nucleic acid sequence encoding RXRG is 3' to a nucleic acid sequence encoding MEF2D.

15. 15. The nucleic acid molecule of any one of claims 1 to 14, wherein the promoter is selected from the group consisting of glial fibrillary acidic protein (GFAP), CAR2, CD44, GLUL, PDGFRA, retinaldehyde binding protein 1 (RLBP1), S100B, SLC1A3, VIM, ProB2, GLAST, CAG and CMV.

16. 16. The nucleic acid molecule of claim 1, wherein the promoter is selected from the group consisting of GFAP, RLBP1, ProB2 and GLAST.

17. 17. A nucleic acid molecule according to any one of claims 1 to 16, wherein the promoter is a macroglia-specific promoter.

18. 18. A nucleic acid molecule according to any one of claims 1 to 17, wherein the macroglia are retinal macroglia.

19. 19. A nucleic acid molecule according to any one of claims 1 to 18, wherein the macroglia are Müller glia and / or astrocytes.

20. 20. A nucleic acid molecule according to any preceding claim, wherein the promoter is a GFAP promoter.

21. 21. A nucleic acid molecule according to any preceding claim, wherein the promoter is the gfaABC1D GFAP promoter.

22. A vector comprising a nucleic acid molecule according to any one of claims 1 to 21.

23. The vector of claim 22 which is a viral vector.

24. 24. The vector of claim 23, wherein the viral vector is selected from the group consisting of a lentiviral vector, a Sendai vector, a herpes simplex virus (HSV) vector, an adenoviral vector, an adeno-associated virus (AAV) vector, an episomal vector and a retroviral vector.

25. 25. The vector of claim 24, wherein the viral vector is selected from the group consisting of a lentiviral vector, an adenoviral vector, and an adeno-associated viral (AAV) vector.

26. The vector of claim 25 , wherein the viral vector is an AAV vector.

27. The vector of claim 25 , wherein the viral vector is a lentiviral vector.

28. The vector of claim 22 , wherein the vector is a non-viral vector.

29. 29. The vector of claim 28, wherein the non-viral vector is selected from the group consisting of a liposome, a nanoparticle, naked DNA, a plasmid and a transposon.

30. The vector of claim 29 , wherein the non-viral vector is a repRNA vector or an mRNA vector.

31. 31. A composition comprising a nucleic acid molecule according to any one of claims 1 to 21 or a vector according to any one of claims 22 to 30, and a pharma- ceutically acceptable carrier.

32. (a) a first nucleic acid molecule according to any one of claims 1 to 21; and (b) a second nucleic acid molecule, the second nucleic acid molecule comprising a promoter operably linked to a nucleic acid sequence encoding one or more transcription factors selected from the group consisting of myocyte enhancer factor 2D (MEF2D), retinoid X receptor gamma (RXRG) and cone-rod homeobox (CRX), or functional variants thereof, or a combination thereof, wherein the promoter is for expression of the one or more transcription factors in macroglia; as a combined preparation for simultaneous, separate or sequential use in the treatment of retinal diseases or retinal degeneration.

33. 33. The article of manufacture of claim 32, wherein at least one of the first nucleic acid molecule and the second nucleic acid molecule encodes MEF2D or a functional variant thereof, and at least one of the first nucleic acid molecule and the second nucleic acid molecule encodes RXRG or a functional variant thereof.

34. 34. The article of manufacture of claim 32 or 33, wherein at least one of the first and second nucleic acid molecules encodes CRX or a functional variant thereof.

35. (a) the first nucleic acid molecule comprises a nucleic acid sequence encoding MEF2C and a nucleic acid sequence encoding RXRG; and / or (b) the second nucleic acid molecule comprises a nucleic acid sequence encoding MEF2D and a nucleic acid sequence encoding CRX; 35. A product according to any one of claims 32 to 34.

36. A method for converting a retinal source cell into a retinal target cell by introducing one or more transcription factors, including MEF2C, or functional variants thereof into the retinal source cell, thereby converting the retinal source cell into the retinal target cell.

37. The method of claim 36, wherein the retinal source cells are macroglia.

38. 38. The method of claim 36 or 37, wherein the retinal source cells are Muller glial cells or astrocytes.

39. 39. The method of any one of claims 36 to 38, wherein the targeted cells of the retina are photoreceptor-like cells or retinal pigment epithelium (RPE)-like cells.

40. 40. The method of claim 39, wherein the photoreceptor-like cells are cone photoreceptor-like cells.

41. The method of any one of claims 36 to 40, wherein one or more transcription factors are introduced via a nucleic acid molecule of any one of claims 1 to 21, a vector of any one of claims 22 to 30, a composition of claim 31, or a product of any one of claims 32 to 35.

42. 42. The method of any one of claims 36 to 41, comprising culturing under suitable conditions for at least 4 days.

43. 43. A cell produced by the method of any one of claims 36 to 42.

44. 44. A nucleic acid molecule according to any one of claims 1 to 21, a vector according to any one of claims 22 to 30, a composition according to claim 31, or a cell according to claim 43, for use in treating a retinal disease or retinal degeneration.

45. A method for treating a retinal disease or retinal degeneration in a subject, comprising administering a therapeutically effective amount of a nucleic acid molecule described in any one of claims 1 to 21, a vector described in any one of claims 22 to 30, a composition described in claim 31, a product described in any one of claims 32 to 35, or a cell described in claim 43 to the retina of a subject in need of treatment.

46. A nucleic acid molecule according to any one of claims 1 to 21, a vector according to any one of claims 22 to 30, a composition according to claim 31, a product according to any one of claims 32 to 35, a cell according to claim 43, or a method of treating a retinal disease or retinal degeneration according to claim 45, wherein the retinal disease or retinal degeneration is age-related macular degeneration (AMD), retinitis pigmentosa (RP), late Best's disease, Stargut macular dystrophy, cone-rod dystrophy or glaucoma.

47. A nucleic acid molecule according to any one of claims 1 to 21, a vector according to any one of claims 22 to 30, a composition according to claim 31, a product according to any one of claims 32 to 35, a cell according to claim 43, or a method of treating a retinal disease or retinal degeneration according to claim 45, wherein AMD is dry AMD.

48. 46. ​​The method of treating a retinal disease or retinal degeneration according to claim 45, comprising administering to said patient a nucleic acid molecule according to any one of claims 1 to 21, a vector according to any one of claims 22 to 30, a composition according to claim 31, a product according to any one of claims 32 to 35, a cell according to claim 43, or a method of treating a retinal disease or retinal degeneration according to claim 45, wherein the dry AMD is late dry AMD.

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