Induced photoreceptor cells and methods for their production

Abstract

The present invention relates to a method for producing induced photoreceptor cells derived from initial cells, the method comprising providing the initial cells with one or more transcription factors (TFs), including at least GON4L. In a preferred embodiment of the present invention, the initial cells are human induced pluripotent stem cells (iPSCs). In another embodiment, the method comprises providing the initial cells with the transcription factors OTX2 and / or NEUROD1. The present invention also relates to cells that can be produced by the method of the present invention, as well as to the use of these cells as a medicament in the treatment of retinopathy, vectors for inducing the photoreceptor cells of the present invention, and combinations of transcription factors for this purpose.

Description

[Technical Field]

[0001] The present invention relates to a method for producing induced photoreceptor cells from an initial cell, the method comprising providing the initial cell with one or more transcription factors (TFs), including at least GON4L. In a preferred embodiment of the present invention, the initial cell is a human induced pluripotent stem cell (iPSC). In another embodiment, the method comprises providing the initial cell with the transcription factors OTX2 and / or NEUROD1. The present invention also relates to cells that can be produced by the method of the present invention, as well as to the use of these cells as medicaments in the treatment of retinopathies, vectors for inducing the photoreceptor cells of the present invention, and combinations of transcription factors for this purpose. [Background technology]

[0002] The use of pluripotent stem cells in regenerative therapy for the treatment of retinal diseases has been discussed in the literature, and several approaches to achieve this goal have been proposed (Non-Patent Document 1, Non-Patent Document 2). Various methods for producing photoreceptor cells have emerged. One method promotes the differentiation of photoreceptors from human embryonic stem cells by adding growth factors, inhibitors, or small molecule compounds (Non-Patent Document 3). In another approach, retinal progenitor cells, such as photoreceptor progenitor cells, are differentiated from mouse iPSCs using manipulation of the Wnt and TGF-β / BMP signaling pathways by using specific inhibitory molecules (Non-Patent Document 4).

[0003] Furthermore, direct cell conversion from somatic cells (1, 2) or stem cells (via 3D organoids) (3-6) has been suggested. Direct conversion from somatic cells utilizes overexpression of transcription factors (TFs) in human fibroblasts to produce photoreceptor-like cells in very small quantities. For example, in Non-Patent Document 5, human photoreceptor cells were induced from fibroblasts by a defined combination of the transcription factors CRX, RAX, OTX2, and NEURD. Another approach is to produce human retinal organoids from human iPSCs, which dissociate after more than 100 days in culture, resulting in the need for large-scale purification of approximately 10% of the photoreceptors.

[0004] Photoreceptors must be enriched from 2D (direct conversion from fibroblasts) or 3D organoids, and all dissociation and purification protocols are technically challenging because they stress the cells and rely on specific markers for fluorescence-activated cell sorting (FACS) or magnetic-activated cell sorting (MACS) (7). Furthermore, the proliferation time of human fibroblasts is long compared to human iPSCs, which is critical for the quantity of the starting cell population. 3D retinal organoids must be cultured for more than 100 days before photoreceptor harvesting, which introduces batch effects and reduces the final quality. Longer incubation times and complex downstream processing further increase the cost of medical products for cell transplantation.

[0005] From extensive studies of in vivo retinogenesis, many transcription factors important for photoreceptor development are known and have been applied to human fibroblasts. However, they are insufficient to promote photoreceptor differentiation from human iPSCs or other pluripotent cells, as in human iPSCs. Photoreceptor progenitor cells have different cellular ground states, and knowledge from fibroblast transdifferentiation protocols cannot be applied to other early cell types, especially iPSCs.

[0006] To transplant human photoreceptors into patients' retinas to treat blindness, an efficient protocol for inducing large quantities of high-quality human photoreceptors from human induced pluripotent stem cells (iPSCs) is required. Therefore, it is necessary to develop a fast, efficient, easily adaptable, homogeneous, and controllable differentiation protocol to provide human photoreceptors of cell therapy quality.

[0007] In light of these prior art techniques, there remains a significant need in the art for fast, efficient, and uniform differentiation protocols for producing induced photoreceptors from primary cells, such as human iPSCs, that provide cell quantity and quality of induced photoreceptors for cell transplantation therapy to replace damaged or degenerated photoreceptors. [Prior art documents] [Non-patent literature]

[0008] [Non-Patent Document 1] Oswald and Baranov, 2018 ..Regenerativemedicine in the retina: from stem cells to cell replacement therapy", Ther Adv Ophthalmol [Non-patent document 2] Weed and Mills, 2017 "Strategies for retinal cell generation from human pluripotent stem cells", Stern Cell Investig. [Non-patent document 3] Zhou et al., 2015 "Differentiation of human embryonic stem cells into cone photoreceptors through simultaneous inhibition of BMP, TGF b and Wnt signaling"Development2015 Oct 1; 142(19):3294-306 [Non-patent document 4] Xie et al. PLOS ONE, vol. 9, no. 11, 2014-11-17, page e112175 [Non-patent document 5] Seko et al. (GENES TO CELLS, vol. 19, no. 3, 2014-01 -24, pages 198-208) Summary of the Invention [Problem to be solved by the invention]

[0009] In light of the prior art, the technical problem underlying the present invention is to provide an alternative or improved method for producing induced photoreceptor cells. Another object of the present invention is to provide an alternative or improved therapeutic agent for treating pathologies associated with damaged or degenerated photoreceptors. In addressing these objectives, the present invention seeks to avoid the disadvantages of the prior art. [Means for solving the problem]

[0010] This problem is solved by the features of the independent claims. Preferred embodiments of the invention are provided by the dependent claims.

[0011] Accordingly, the present invention relates to a method for producing derived photoreceptor cells from initial cells, the method comprising providing the initial cells with one or more transcription factors (TFs), including at least GON4L.

[0012] It was quite surprising that the transcription factor GON4L, which has never been described in the context of photoreceptors, is an effective factor for inducing the photoreceptor phenotype in early cells, reprogramming them to photoreceptor-like cells. Only by performing an unbiased library screen including virtually all human transcription factors could we identify this completely unexpected transcription factor. Surprisingly, it was not sufficient to induce the differentiation of early cells into photoreceptor-like cells or photoreceptor precursor cells using transcription factors already known to be involved in photoreceptor development; GON4L was required to achieve this. Importantly, the method of the present invention allows for the rapid and efficient induction of the photoreceptor phenotype in early cells, resulting in a relatively homogeneous cell population that can, optionally, be further purified by isolating the induced photoreceptor cells.

[0013] In contrast to known 2D cell culture protocols for producing photoreceptor cells from initial cells, the method of the present invention is fast and applicable to a variety of cell types, including proliferating cells such as iPSCs. The supply of the transcription factor GON4L and potentially additional transcription factors can be carried out in stages. For example, it is possible to deliver one or more exogenous nucleic acids encoding the necessary transcription factors to initial cells without inducing their expression from the exogenous nucleic acids. The initial cells can then be propagated for several rounds of replication before inducing the expression of the factors from the nucleic acids, corresponding to the supply of transcription factors. This allows for significant expansion of the initial cells before inducing photoreceptor differentiation, enabling the production of large numbers of induced photoreceptor cells from a small number of initial cells. This represents a significant advantage over known 2D differentiation protocols, which use, for example, slowly dividing fibroblasts as initial cells.

[0014] Furthermore, cells exhibiting a phenotype resembling photoreceptor precursors can be identified in culture systems very early after the provision of GON4L and potentially additional transcription factors. Such early stage progenitor cells, as well as cells corresponding to more differentiated or mature photoreceptor developmental stages, can be readily isolated by means described herein and known in the art for downstream application of the cells.

[0015] The provision of GON4L, potentially other transcription factors, particularly OTX2 and NEUROD1, and / or in combination with other factors represents a novel method for inducing a photoreceptor phenotype in early cells in culture.

[0016] Without being limited by theory, it is believed that the use of GON4L to induce the photoreceptor phenotype is necessary to prime early cells for photoreceptor differentiation.

[0017] It was quite surprising that the provision of GON4L was sufficient to induce the photoreceptor phenotype in early cells, particularly when iPSCs were used as the initial cells. As is evident from the accompanying examples, GON4L is the only transcription factor required to induce expression of a reporter gene under the control of a photoreceptor-specific promoter, although expression in combination with other transcription factors is preferred.

[0018] According to the present invention, expression of GON4L appears to be necessary to achieve the technical effect of inducing photoreceptor cells from early cells, and is preferably used in combination with the expression of another transcription factor to induce the photoreceptor phenotype, more preferably via the combined expression of GON4L with OTX2 and / or NEUROD1 (see Figures 5 and 6).

[0019] A major advantage of the present invention is that it allows for the production of induced photoreceptor cells with high purity, which simplifies further downstream processing to purify and enrich the cells into a homogenous population.

[0020] In an embodiment of the present invention, the initial cells are pluripotent or multipotent mammalian cells, which are differentiated into induced photoreceptor cells by providing the initial cells with one or more transcription factors (TFs), including at least GON4L.

[0021] Preferably, the initial cells are induced pluripotent stem cells (iPSCs).

[0022] The use of iPSCs as initial cells for the methods of the present invention is particularly advantageous because these cells can be easily expanded due to their proliferative capacity. That is, in embodiments in which one or more transcription factors are provided in an inducible manner via expression from one or more nucleic acids, it is possible to proliferate iPSCs after delivery of the nucleic acids but before inducing transcription factor expression from the nucleic acids. Thus, it is possible to derive large numbers of photoreceptor cells from a small number of initial cells. This advantage also applies to other proliferative or proliferative cells that may serve as initial cells. Furthermore, it is possible to generate iPSCs from individual patients as initial cells for the methods of the present invention. Such individual cells can be used as a drug in the treatment of the same patient after induction of the photoreceptor phenotype according to the present invention. That is, it is possible to generate large numbers of patient-specific induced photoreceptor cells from only a small number of isolated patient-specific cells.

[0023] In a preferred embodiment of the invention, the initial cells are of human origin.

[0024] Initial cells of human origin are preferred and particularly advantageous for therapeutic and research applications of the photoreceptor cells of the present invention because the induced photoreceptor cells generated from such cells are also human. When the induced photoreceptors are of human origin, they can be used for transplantation into patients in need of such cells, such as patients suffering from retinal degeneration or other ocular diseases. Furthermore, the use of human cells is a significant advantage for using the cells of the present invention for research and development purposes, such as drug screening and development.

[0025] In a specific embodiment, the initial cells are fibroblasts. Fibroblasts are advantageous initial cells because they are easily accessible from donors and easy to culture. That is, it is possible to generate a large number of fibroblasts from patients that can be immediately used as initial cells in the method of the present invention, resulting in the rapid generation of induced photoreceptor cells after isolation of the cells from the patient. More preferred initial cells may be bone marrow-derived cells such as hematopoietic stem cells, proliferative progenitor cells present in bone marrow, leukocytes, and lymphocytes.

[0026] In further embodiments, other somatic cells or progenitor cells can be used as initial cells.Those skilled in the art can select appropriate initial cells taking into account general knowledge and the latest technology.It has been described that specific cell phenotypes of differentiated cells can be derived from stem cells such as iPSCs, or from other initial cells such as fibroblasts or other somatic cells that are fully differentiated or still have the potential to differentiate.From such studies, those skilled in the art can conclude that transcription factors that can promote the induction of specific phenotypes in iPSCs as initial cells are also useful for inducing such specific phenotypes in different cell types used as initial cells.

[0027] The induction of human neural cells by defined transcription factor expression has previously been described, whereby somatic cell nuclear transfer, cell fusion, or expression of lineage-specific factors has been shown to induce cell fate changes in diverse somatic cell types (Pang et al., Nature. 2011 May 26;476(7359):220-3). For example, forced expression of different combinations of three transcription factors (Brn2, AscM, and Mytl) can efficiently convert mouse fibroblasts and pluripotent cells into functionally induced neuronal (iN) cells.

[0028] That is, although the use of iPSCs as an initial cell type for the present invention is a preferred embodiment, one skilled in the art would not expect the use of iPSCs as an initial cell to be an essential feature of the present invention. In contrast, the fact that GON4L can induce a photoreceptor phenotype in iPSCs indicates that GON4L can also promote the induction of a photoreceptor phenotype in other initial cell types.

[0029] In one embodiment, the initial cell is not an embryonic stem cell or other cell obtained from an embryo.

[0030] In an embodiment of the present invention, the induced photoreceptor cells are cone cells.

[0031] In a further embodiment, the induced photoreceptor cells are rod cells.

[0032] In some embodiments, the induced photoreceptor cells are photosensitive retinal ganglion cells.

[0033] A major advantage of the methods of the present invention is that by modifying the culture conditions to supply a combination of factors, it becomes possible to generate rod, cone, or photosensitive retinal ganglion cells directly from the initial cells, which is particularly advantageous when using the derived photoreceptor cells in downstream applications specific to a particular photoreceptor subtype.

[0034] In an embodiment of the invention, the method of the invention comprises providing the initial cell with one or more transcription factors selected from CRX, NEUROD1, NR2E1, NR2E3, NRL1, OTX2, ONECUT1, PAX6, RAX, RORB, RXRG, SIX3, SIX6, SOX2, THRB and VSX2.

[0035] In an embodiment of the present invention, one or more transcription factors including GON4L are provided to the initial cell. In another embodiment, three or more transcription factors including GON4L are provided to the initial cell.

[0036] In an embodiment of the present invention, the method comprises providing two or more transcription factors including GON4L and one or more transcription factors selected from CRX, NEUROD1, NR2E1, NR2E3, NRL1, OTX2, ONECUT1, PAX6, RAX, RORB, RXRG, SIX3, SIX6, SOX2, THRB and VSX2 to the initial cells.

[0037] It was quite surprising that providing GON4L together with at least one additional transcription factor, preferably two, was effective in inducing a photoreceptor phenotype in early cells such as iPSCs. The accompanying examples demonstrate that combining GON4L with SIX6, NEUROD1, or OTX2 produced the desired effect. In one embodiment, the invention involves providing GON4L and OTX2 to early cells, such as, preferably, iPSCs. In one embodiment, the invention involves providing GON4L and SIX6 to early cells, such as, preferably, iPSCs. In one embodiment, the invention involves providing GON4L and NEUROD1 to early cells, such as, preferably, iPSCs.

[0038] These transcription factors, when supplied in combination with GON4L, have been shown to promote photoreceptor development from early cells and more efficiently induce the photoreceptor phenotype in early cells.

[0039] In a preferred embodiment, the methods of the invention involve providing OTX2 and / or NEUROD1 to the initial cells.

[0040] Surprisingly, expression of either OTX2 or NEUROD1, and especially both transcription factors, was found to improve the differentiation capacity of early cells into induced photoreceptors when supplied in combination with GON4L.

[0041] In one embodiment, a combination of GON4L, OTX2, and NEUROD1 is provided to initial cells. In one embodiment, the present invention includes providing GON4L, OTX2, and NEUROD1 to iPSCs as initial cells. In some embodiments, transcription factors are provided approximately simultaneously to induce a photoreceptor phenotype in initial cells using the methods of the present invention. In further embodiments, transcription factors may be provided sequentially. For example, GON4L may be provided minutes, hours, or days before a second transcription factor, such as OTX2 and / or NEUROD1. A third transcription factor may be provided simultaneously with or at a later time than the first or second transcription factor. In an embodiment of the present invention, GON4L is provided after at least one other transcription factor, such as OTX2 and / or NEUROD1. This sequential provision also applies to other factors, such as additional transcription factors or microRNAs, that may be provided to initial cells using the methods of the present invention.

[0042] In an embodiment of the present invention, OTX2, NEUROD1 and GON4L are provided to the primary cells essentially simultaneously or sequentially.

[0043] The order of supply may be (i) GON4L, (ii) OTX2, and (iii) NEUROD1, or (i) GON4L, (ii) NEUROD1, and (iii) OTX2. Further, the order of supply may be (i) OTX2, (ii) NEUROD1, and (iii) GON4L, or (i) OTX2, (ii) GON4L, and (iii) NEUROD1. Also, the order of supply may be (i) NEUROD1, (ii) GON4L, and (iii) OTX2, or (i) NEUROD1, (ii) OTX2, and (iii) GON4L.

[0044] Alternatively, one of the factors may be provided first before the other two factors are provided at approximately the same time, for example, GON4L before OTX2 and NEURD1, or OTX2 before GON4L and NEUROD1, or NEUROD1 before GON4L and OTX2.

[0045] The time frame between the provision of first, second, third, and / or additional transcription or other factors that may be provided in connection with the methods of the invention may be in the range of about 10, 15, 20, 25, 30, 40, 50, and / or 60 minutes. It may also be in the range of about 1, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, and / or 24 hours. In embodiments of the invention, the time frame between the provision of transcription and / or other factors in connection with the methods of the invention may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and / or 20 days.

[0046] In an embodiment of the invention, the method of the invention comprises providing a microRNA, preferably miR-182 and / or miR-183, to the initial cells.

[0047] In a further embodiment, the one or more transcription factors and / or one or more microRNAs (e.g., miR-182 and / or miR-183) are expressed from one or more exogenous nucleic acid molecules in the initial cell, wherein expression from the exogenous nucleic acid preferably results in a higher level than is present in the initial cell, e.g., human iPSC.

[0048] In another embodiment of the invention, the initial cells are supplied with one or more transcription factors and other factors, such as miR-182 and / or miR-183, for at least 4 days, preferably about 7-10 days. In an embodiment of the invention, supplying one or more transcription factors and potentially other factors for only about 1 day is sufficient to induce reprogramming of the initial cells into induced photoreceptor cells, even though the photoreceptor phenotype may only arise after an additional time frame.

[0049] Even if the initial exogenous supply of one or more transcription factors occurs only for a short period of time, such as one day, supplying GON4L and potentially other factors (e.g., OTX2 and NEUROD1) for only a short initial time, such as one day, may be sufficient to induce a transdifferentiation program in early cells to develop into induced photoreceptor cells. In embodiments of the present invention, early cells are supplied with one or more transcription factors and potentially other factors (e.g., miR-182 and / or miR-183) for at least 0.25, 0.5, 0.75, 1.5, 2, 2.5, 3, 3.5, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 days. Different factors supplied during the method of the present invention can be supplied for different periods of time or sequentially.

[0050] In embodiments of the invention, one or more transcription factors and potential microRNAs (e.g., miR-182 and / or miR-183) are expressed in the primary cells from one or more viral vectors, preferably lentiviral vectors.

[0051] In further embodiments, one or more transcription factors and potential microRNAs (e.g., miR-182 and / or miR-183) are supplied by microinjection, transfection, electroporation of the factors, and / or by transfection or electroporation of exogenous nucleic acid molecules for expression of the factors, e.g., mRNA molecules.

[0052] In some embodiments, one or more transcription factors and potential microRNAs (e.g., miR-182 and / or miR-183) are supplied by the PiggyBac (PB) transposon system or other transposon systems. Such transposon systems are advantageous because they provide a safe method of factor delivery to the initial cell, as the genetic elements can be removed from the cell after, for example, transient expression of one or more transcription factors.

[0053] In preferred embodiments, one or more transcription factors and potential microRNAs (eg, miR-182 and / or miR-183) are transiently expressed and / or expression is induced in the primary cells.

[0054] The transient and / or induced expression or expression of factors is particularly advantageous because, after transient and / or induced expression or supply of factors and differentiation of induced photoreceptor cells or induction of a differentiation program leading to the generation of induced photoreceptor cells, the supply of exogenous factors can be terminated, and the photoreceptor phenotype of the cells can be maintained by expression of endogenous factors and / or factors supplied by the cellular environment. After removal of the supplied factors from induced photoreceptor cells, these cells may behave more physiologically because they are no longer forced to be supplied with exogenous factors. Thus, after factor removal, the cells may resemble naturally occurring photoreceptor cells more closely.

[0055] In an embodiment of the present invention, the inducible expression is mediated by tetracycline-dependent transcriptional regulation. Tetracycline or one of its derivatives, such as doxycycline, can be easily supplied to and removed from the initial cell to regulate the expression of transcription factors from exogenous nucleic acid molecules, so the expression of one or more transcription factors and potential microRNAs (e.g., miR-182 and / or miR-183) through tetracycline-regulated transcriptional activation is advantageous.

[0056] In a further embodiment, the method of the present invention comprises administering a cell cycle inhibitor to the initial cells, preferably AraC. Inhibitors such as cell cycle inhibitors are considered to be factors that can be supplied to cells during the method of the present invention. Such inhibitors may simply be added to the cell culture medium at specific times during the method of the present invention. For such inhibitors, the same time frames and criteria as those for continuous supply and supply are applicable, as outlined above for transcription factors and potential microRNAs. The use of cell cycle inhibitors during the method of the present invention can be particularly advantageous when supplied after one or more transcription factors that initiate the reprogramming of the initial cells.

[0057] In some embodiments, the cell cycle inhibitor is administered to the initial cells after providing the one or more transcription factors, preferably 5 days after providing the one or more transcription factors, hi some embodiments, the cell cycle inhibitor is administered to the initial cells 0.25, 0.5, 0.75, 1.5, 2, 2.5, 3, 3.5, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 days after providing the one or more transcription factors.

[0058] In an embodiment of the method of the present invention, the initial cells are cultured on a basement membrane-like matrix, e.g., Matrigel, or on another gelatinous protein mixture, such as specific collagen or laminin molecules that support the development or maintenance of photoreceptor cells, such as poly-L-lysine or poly-D-lysine.

[0059] In some embodiments, the methods of the invention involve co-culturing the early cells with retinal pigment epithelial cells (RPE cells). Such methods of the invention are particularly advantageous because the RPE cells provide a cellular environment that promotes the differentiation of the early cells into induced photoreceptor cells.

[0060] Aspects Related to Detection of Induced Photoreceptor Cells

[0061] In embodiments of the methods of the present invention, the induced photoreceptor cells produced from the initial cells are determined by the photoreceptor reporter system present in the initial cells, preferably said photoreceptor reporter system comprising one or more photoreceptor-specific promoter sequences, such as sequences from arrestin and / or rhodopsin promoters, and one or more reporter genes, such as fluorescent protein genes, and / or selectable markers.

[0062] The use of photoreceptor reporter systems in the present invention is advantageous because they can indicate the development of a photoreceptor phenotype in early cells, thus providing guidance on which induced cells can be used for downstream applications. Furthermore, the use of fluorescent reporter genes, such as GFP, RFP, and dsRed, allows for the detection of photoreceptor phenotypes by a variety of methods, including microscopy and flow cytometry. The use of several different promoter sequences with different specificities further enables phenotyping. For example, the simultaneous use of cone- and rod-specific promoter sequences with different reporter genes, such as genes encoding fluorescent proteins of different colors, allows for the detection and subsequent isolation of rod- and cone-like cells in mixed cultures in the present methods. For example, because rhodopsin is a rod-specific protein, activity of the rhodopsin promoter indicates the development of a rod phenotype. In contrast, certain arrestin proteins, such as arrestin-3, are cone-specific, and their promoters can be used in the present invention to monitor cone development. Additionally, selectable markers, such as genes that render cells resistant to certain toxic chemicals, such as antibiotics, can be expressed under the control of photoreceptor-specific promoters to select for photoreceptors derived from mixed cell cultures.

[0063] In an embodiment of the present invention, induced photoreceptor cells are isolated from a cell culture that may contain uninduced initial cells or cells other than induced photoreceptors. Isolation of induced photoreceptors can occur through the use of proteins such as marker genes or fluorescent proteins, for example, through fluorescence-activated cell sorting. Isolation of induced photoreceptor cells can also occur through magnetic cell separation (FACS) sorting, for example, based on the expression of surface markers. Furthermore, isolation can be achieved through the expression of selectable markers that make cells resistant to compounds, thereby eliminating all uninduced cells from the culture. Those skilled in the art can use additional techniques known in the art to isolate induced photoreceptor cells from cell culture systems containing additional cell types.

[0064] In a further aspect of the invention, it may be useful to generate monoclonal or clonal cell lines from the induced photoreceptor cells.

[0065] In some embodiments of the present invention, the generation of induced photoreceptor cells is determined by the expression of genes or proteins that are specifically expressed in photoreceptor cells but not in the initial cells of the present method. In some embodiments, the marker molecules described below are more highly expressed in induced photoreceptors than in initial cells such as iPSCs. In some embodiments of the present invention, the generation of induced photoreceptor cells is determined by the expression of endogenous recoverin, NCAM, OTX, CRX, RCVRN, RHO, OPN1 SW, and / or OPN1 LW. Expression detection can occur at the protein or mRNA level, for example, by qPCR, antibody-mediated detection methods, and other well-established techniques known to those skilled in the art.

[0066] In some embodiments, the generation of induced photoreceptor cells is determined by the formation of neurite outgrowth in an in vitro assay, which is an indicator of a neuronal phenotype indicative of the induction of neuroepithelial photoreceptors.

[0067] In some embodiments, the initial cells are iPSCs and the generation of induced photoreceptor cells is determined by the loss of Tra1-60 expression, an iPSC marker that disappears from initial cells once the photoreceptor phenotype is induced.

[0068] Induced Photoreceptor Cell Aspects

[0069] The present invention further relates to induced photoreceptor cells produced by the methods of the present invention.

[0070] Furthermore, the present invention relates to induced photoreceptor cells obtained by the methods of the present invention, i.e., all types of induced photoreceptor cells that exhibit the same phenotype (e.g., specific gene expression profile, surface marker combination, cell shape and / or cell function) as the cells produced by the methods of the present invention, which differs in morphology from naturally occurring photoreceptor cells and from induced photoreceptor cells produced by state-of-the-art methods.

[0071] For example, because the methods of the present invention can be carried out using different means of providing the transcription factors of the present invention, the cells produced by the methods of the present invention may not necessarily be best described by their structural characteristics. For example, transcription factors may be provided by exogenous nucleic acids. However, these nucleic acids may later disappear from the cells (be transient). That is, the cells are not necessarily defined by the presence of exogenous nucleic acids. Similarly, with respect to the methods of the present invention, after providing the transcription factors, the cells may begin to express endogenous transcription factors, and after a certain period of time, the exogenous transcription factors may no longer be detectable while the cells maintain the photoreceptor phenotype induced by the expression of the endogenous factors. However, one skilled in the art can analyze the cells of the present invention produced by the methods of the present invention to distinguish them from physiologically existing photoreceptor cells, for example, by analyzing their overall expression profile.

[0072] In some embodiments, the methods described herein induce photoreceptor cells that exhibit the characteristics of induced photoreceptors described in the Examples. The present invention further relates to the induced photoreceptor cells described in the Examples disclosed herein. The cells of the present invention express at least one of the photoreceptor markers measured in the Examples, including NCAM, OTX2, CRX, RCVRN, RHO, mCAR, OPN I SW, and OPN I LW. In some embodiments, the induced photoreceptors of the present invention do not express SOX2 or OCT4. In this regard, in some embodiments, the expression of each factor can be determined by comparing the expression in the cells of the present invention with the expression level of each factor in iPSCs. For example, in some embodiments, the absence of OCT4 or SOX2 can be associated with an expression level that is about 10% or less than that in iPSCs. On the other hand, in some embodiments, the expression of photoreceptor markers in the cells of the present invention can be about 10-fold higher than that in iPSCs.

[0073] The present invention further relates to induced photoreceptor cells, which preferably comprise at least one exogenous nucleic acid molecule comprising a sequence encoding GON4L, under the control of suitable regulatory nucleic acid sequences, such as, for example, a constitutive or inducible promoter or promoter / enhancer combination.The cells of the present invention can be produced using the kits of the present invention disclosed herein.Generally, the features disclosed in relation to the methods or kits of the present invention are also relevant and disclosed herein in relation to the cells of the present invention, and vice versa.

[0074] The cells of the present invention can be used for research and development purposes, for example, to identify, test and screen potential drugs that affect or act on photoreceptor cells.

[0075] Furthermore, the present invention relates to the cells of the present invention for use as a pharmaceutical in the treatment of retinopathies, such as retinal degeneration. The cells can be transplanted into the retina of an affected patient. The transplanted cells of the present invention can be at different differentiation stages. For example, induced photoreceptor cells can be at the photoreceptor precursor stage at the time of transplantation and develop into mature photoreceptors after transplantation into the retina. Alternatively or additionally, more mature or mature induced photoreceptor cells can be transplanted, although this may depend on the needs and condition of the individual patient.

[0076] A major advantage of the method of the present invention is that it allows for the provision of patient-specific induced photoreceptor cells that can be used as a medicine in patient treatment. Furthermore, the use of HLA-matched iPSCs from an iPSC bank allows for the provision of appropriate induced photoreceptor cells generated by matching iPSCs to the patient. This is particularly advantageous when the condition leading to the need for induced photoreceptor cell transplantation or treatment is genetic. This is because it is possible to provide matched photoreceptor cells without relying on the patient's own cell donation, which may require correction of the genetic cause leading to the disease requiring photoreceptor transplantation.

[0077] Further aspects of the invention

[0078] The present invention also relates to a kit for producing photoreceptor cells derived from initial cells according to the methods disclosed herein. The kit of the present invention comprises: a. A vector system for delivering GON4L and optionally further transcription factors (preferably OTX2 and / or NEUROD1) to the initial cells, and optionally miR-182 and / or miR-183; b. Reagents for detecting induced photoreceptor cells generated from initial cells; for example, i. Photoreceptor-specific reporter system ii. antibodies to detect photoreceptor marker proteins, such as OPN1 SW, OPN1 LW, recoverin, and / or NCAM, and / or iii. Primers for detecting OTX, CRX, RCVRN, RHO, OPN1 SW, OPN1 MW and / or OPN 1 LW mRNA by PCR, and c. Optionally, a cell cycle inhibitor, preferably AraC.

[0079] Furthermore, the present invention relates to an expression vector system comprising one or more nucleic acid sequences operably linked to one or more promoters, said sequences encoding one or more transcription factors (TFs), including at least GON4L, OTX2, and NEUROD1, and optionally miR-182 and / or miR-183. Preferred embodiments of the vector are as follows:

[0080] The present invention also relates to combinations of transcription factors, including at least GON4L, OTX2, and NEUROD1, which may involve combinations of transcription factors in protein form, combinations of nucleic acids encoding the transcription factors, combinations of nucleic acids encoding the transcription factors in vectors or other expressible forms, or combinations of these transcription factors in modified cells above the levels in initial cells, such as iPSCs. [Brief explanation of the drawings]

[0081] The present invention is further illustrated by the following figures, which are not intended to limit the scope of the invention but represent preferred embodiments or aspects of the invention provided for the purpose of further explanation.

[0082] [Figure 1]In vitro photoreceptor differentiation from hiPSCs by transcription factor overexpression. (A) Schematic diagram of the cone reporter cassette introduced into human iPSCs. GFP is under the cone arrestin promoter and is active only in cone photoreceptors. (B) Schematic diagram of the cone differentiation protocol. The transcription factor is under the doxycycline (DOX)-inducible promoter pTRE. In the presence of DOX, DOX binds to the transactivator rtTA3 and initiates expression of the transcription factor. Less than 10 days of DOX treatment is sufficient to obtain cone photoreceptors in 2D culture. Scale bar, 50 pm. [Figure 2] Flow cytometry analysis of overexpression of the transcription factor combination OTX2, NEUROD1, and GON4L in human iPSCs. Overexpression of the transcription factor combination OTX2, NEUROD1, and GON4L for 10 days differentiates human induced pluripotent stem cells into 26.1% cone photoreceptor-like cells. Treating them with the cell cycle inhibitor AraC on day 5 removes the pool of proliferating progenitor cells, increasing differentiation efficiency to 51.6% (average, n=3). [Figure 3] Microscopic analysis of the overexpression of the transcription factor combination OTX2, NEUROD1, and GON4L in human iPSCs. Figures S37 and S36. Overexpression of the transcription factor combination OTX2, NEUROD1, and GON4L (ONG) in human induced pluripotent stem cells for 7 days in the presence of doxycycline (+DOX) leads to the upregulation of photoreceptor-specific markers. GFP-positive cells (driven by the cone arrestin promoter) coexpress the photoreceptor precursor marker recoverin (RCVRN, red), indicating differentiation into cone photoreceptor-like cells. In our protocol, cells are cultured on Matrigel (a protein mixture secreted by mouse sarcoma cells), but photoreceptor-specific laminin may be required to obtain improved photoreceptor-specific cell morphology. Nevertheless, using the current culture conditions, neurite outgrowth, a core function of developing neurons, was observed. [Figure 4]Schematic of generating induced photoreceptors via transcription factor induction. A selected set (left) or library of transcription factors (right) were induced in human iPS cell lines harboring photoreceptor (PR)-specific fluorescent reporters. Detailed analysis and comparison of in vivo PRs allows for sophisticated evaluation of the generated induced PRs. [Figure 5] Fluorescence-activated cell sorting plot of induced photoreceptor cells. Of 87 fluorescent cells, 85 showed green fluorescence and 2 showed red fluorescence. [Figure 6] Photoreceptor-specific gene profiles of fluorescent cells expressing GON4L. All GON4L-expressing cells were positive for the photoreceptor precursor and progenitor markers CRX and OTX2, eight cells were positive for the pan-photoreceptor marker RCVRN, and one FACS-sorted cell was positive for the late cone marker OPN1 SW. Cells were found to co-express transcription factors distinct from the biased group, six of which co-expressed OTX2 and three co-expressed NEUROD1. [Figure 7] PGP1 cR-ONG cells downregulate stem cell markers (A) and upregulate photoreceptor-specific markers (B) as analyzed by qPCR. PGP1 cR-ONG cells downregulate stem cell markers (A) and upregulate photoreceptor-specific markers (B) during the 10-day differentiation protocol. (A) The stem cell markers OCT4 and SOX2 are significantly downregulated already 3 days after overexpression of OTX2, NEUROD1, and GON4L, indicating that most cells are out of the cell cycle. After 10 days of DOX treatment, or 8 days after treatment with the cell cycle inhibitor AraC, the expression levels of the stem cell markers are barely detectable, and the cells are fully differentiated. (B) The photoreceptor markers CRX and RCVRN are upregulated during the course of differentiation, reaching a peak expression at day 10. When AraC is added, the remaining cells upregulate the late photoreceptor marker RCVRN at the expense of the early photoreceptor marker CRX. [Figure 8]Flow cytometry analysis of Tra1-60- / NCAM+ / GFP+ cells after 5 days of overexpression of OTX2-NEUROD1 (ON) or OTX2-NEUROD1-GON4L (ONG) in PGP1 and CRTD5 cone reporter hiPSC lines. The transcription factor combination OTX2-NEUROD1 results in GFP expression from the cone arrestin promoter in approximately 10% of differentiated cells. In the presence of GON4L, photoreceptor differentiation efficiency increases to approximately 25%. The same differentiation efficiency can be reproduced with another cone reporter hiPSC line, CRTD5cR cells. [Figure 9] Overexpression of the transcription factor combination OTX2-NEUROD1-GON4L in hiPSC lines leads to downregulation of pluripotency markers and upregulation of photoreceptor-specific markers, as analyzed by qPCR. Overexpression of the transcription factor combination OTX2-NEUROD1-GON4L for 10 days leads to downregulation of pluripotency markers OCT4 and SOX2 (A) and upregulation of photoreceptor-specific markers RCVRN and CRX (B) in both PGP1 and CRTD5 cone reporter hiPSC lines. Expression levels of the pluripotency marker and late photoreceptor marker RCVRN are comparable between the two hiPSC lines. [Figure 10] Overexpression of the transcription factor combination OTX2-NEUROD1-GON4L leads to the upregulation of photoreceptor-specific markers, as analyzed by immunofluorescence. Overexpression of the transcription factor combination OTX2-NEUROD1-GON4L in PGP1 and CRTD5 cone reporter hiPSCs for 10 days leads to the upregulation of photoreceptor-specific markers. GFP-positive cells (driven by the cone arrestin promoter) co-express the photoreceptor precursor marker CRX (magenta), indicating differentiation into cone photoreceptor-like cells. Neurite outgrowth, a key characteristic of developing neurons, was observed. [Figure 11-1]A 10-day differentiation protocol for obtaining cells for transplantation experiments. (A) Cells are seeded in mTeSR using ROCKi. The next day, DOX is added to initiate OTX2-NEUROD1-GON4L overexpression and photoreceptor differentiation. The cell cycle inhibitor AraC is added from 5 to 7 dpi to remove any potentially proliferating cells. At 10 dpi, cells are collected using a papain dissociation kit (Worthington Biochemical Corporation) and (B) FACS sorted for live (7-AAD-) GFP+ cells. Next, 150,000 cells are transplanted subretinally according to a published protocol (Santos-Ferreira et al. Daylight vision repair by cell transplantation. Stem Cells. 2015Jan;33(1):79-90. doi: 10.1002 / stem.1824). [Figure 11-2] Same as above DETAILED DESCRIPTION OF THE INVENTION

[0083] All documents cited, both patent and non-patent literature, are incorporated herein by reference in their entirety.

[0084] The present invention is directed to a method for producing derived photoreceptor cells from initial cells, the method comprising providing the initial cells with one or more transcription factors (TFs), including at least GON4L.

[0085] In the present invention, the term "induced photoreceptor cells" refers to cells having a phenotype similar to naturally occurring photoreceptor cells or precursor cells of such photoreceptors, which have developed or differentiated from an initial cell that is not a photoreceptor cell. Induced photoreceptor cells exhibit at least a combination of characteristics of photoreceptor cells and photoreceptor precursor cells, such as expression of one or more genes and proteins specific to photoreceptors and their precursor cells, and / or exhibit a photoreceptor-like morphology, including neurite outgrowth. Such markers include, but are not limited to, recoverin (RCVRN), rhodopsin, cone arrestin (arrestin-4), arrestin-1, NCAM, CRX, NEUROD1, NR2E1, NR2E3, NRL1, OTX2, ONECUT1, PAX6, RAX, RORB, RXRG, SIX3, SIX6, SOX2, THRB, VSX2, OTX, RHO, OPN1 SW, OPN1 MW, and / or OPN1 LW. While expression of such markers may be present to some degree in other cell types, such markers may be well known for their involvement in photoreceptor differentiation. Furthermore, the development or induction of photoreceptor cells from initial cells can be monitored or detected by the absence of the initial cell marker. Absolute absence of such a suppressed marker in the initial cells is not required for "suppression" according to the present invention; it may remain at a low level in the cells. Suppression of an initial cell marker can be characterized as a decrease in expression level compared to the initial cells. A reduced level compared to an appropriate control can be used to determine "suppression." Similarly, "activation" of gene expression of photoreceptor-specific genes can be determined by comparison with an appropriate control, such as the respective initial cells. Induced photoreceptors can be characterized by their transcriptional profile, which can be derived from bulk population or single-cell RNA sequencing analysis. Such a profile can be used to distinguish the induced photoreceptor cells of the present invention from naturally occurring photoreceptor cells.

[0086] Photoreceptor cells are a specialized type of neuroepithelial cell found in the retina that are capable of visual phototransduction. Photoreceptors convert light (visible electromagnetic radiation) into signals that can stimulate biological processes. Photoreceptor proteins in these cells absorb photons, causing changes in the cell's membrane potential. Mammalian photoreceptor cells include rods, cones, and light-sensitive retinal ganglion cells. The two classical photoreceptor cells are rods and cones. Rods are thinner than cones and are distributed differently throughout the retina, but their respective chemical processes supporting phototransduction are similar. Photosensitive ganglion cells do not directly contribute to vision but are thought to support circadian rhythms and the pupillary reflex.

[0087] Rods are highly sensitive and can be triggered by a single photon. At very low light levels, visual experience is based solely on rod signals, making it impossible to see color. Cones require very bright light (i.e., more photons) to generate a signal. In humans, there are three different types of cone cells, distinguished by their patterns of response to different wavelengths of light. Color experience is calculated from these three distinct signals. The three types of cone cells respond (roughly) to short-, medium-, and long-wavelength light. The human retina contains approximately 120 million rod cells and 6 million cone cells. The number and ratio of rods to cones varies by species, depending on whether the animal is primarily diurnal or nocturnal. In addition to light-sensitive rods and cones, the human visual system contains approximately 2.4 to 3 million ganglion cells, of which 1 to 2% are photosensitive. The axons of ganglion cells form the two optic nerves.

[0088] The method of the present invention relates to providing one or more transcription factors.In the present invention, providing transcription factors or other factors, such as microRNA, relates to providing, making available, contacting with initial cells, introducing transcription factors into initial cells, or generating transcription factors from within or in the immediate vicinity of initial cells.Transcription factors can be provided at the protein level or in the form of nucleic acids encoding transcription factors.

[0089] That is, when exogenous nucleic acid molecules encoding transcription factors are delivered, the transcription factors are provided during the protein expression from exogenous nucleic acid molecules.Transcription factors can be provided through the expression from any given nucleic acid molecule.This includes the activation of the expression of each transcription factor from endogenous or exogenous nucleic acid molecules.In addition, transcription factors can be delivered directly to cells, for example, by protein transfection.Preferably, the expression of transcription factors occurs in a greater amount than in initial cells (e.g., iPSCs).

[0090] The supply of transcription factors can occur through expression from nucleic acid molecules, such as exogenous nucleic acid molecules. As used herein, "nucleic acid" refers to any nucleic acid molecule, including, but not limited to, DNA, RNA, and hybrids or modified variants thereof. "Exogenous nucleic acid" or "exogenous genetic element" refers to any nucleic acid introduced into a cell that is not a component of the cell's "original" or "natural" genome. The exogenous nucleic acid may or may not be integrated into the genetic material of the target mesenchymal stem cell, or may relate to a stably transduced nucleic acid. Delivery of exogenous nucleic acid can result in genetic modification of the initial cell through permanent integration of the exogenous nucleic acid molecule in the initial cell. However, delivery of exogenous nucleic acid can be transient, meaning that the genetic material delivered to supply one or more transcription factors disappears from the cell after a certain period of time.

[0091] Delivery of nucleic acid molecules and potential genetic modifications of initial cells (e.g., mammalian or human cells, preferably human iPSCs) can be performed and determined by those skilled in the art using commonly available techniques. For example, sequencing of the genome of the initial cells or portions thereof is possible to detect genetic modifications, thereby identifying whether exogenous nucleic acid is present. Alternatively, other molecular biology techniques, such as polymerase chain reaction (PCR), can be applied to identify / amplify exogenous genetic material. Exogenous nucleic acid can be detected by vector sequences or portions of vector sequences remaining at the site of genetic modification. Even if vector sequences (e.g., vector sequences flanking a therapeutic transgene) can be removed from the genome, the addition of the therapeutic transgene may be detected through sequencing efforts by detecting genomic sequences incorporating the therapeutic gene at a "non-native" genomic location.

[0092] Any given gene delivery method for delivering nucleic acid molecules is encompassed by the present invention, preferably viral or non-viral vectors, as well as biological or chemical methods of transfection. This method can result in stable or transient gene expression in the system used. Furthermore, any method known to those skilled in the art for delivering proteins to mammalian cells is encompassed by the present invention, including the delivery of one or more transcription factors and / or microRNAs or other factors. All known methods for delivering nucleic acid molecules and proteins, as well as other biological and chemical molecules that can act as factors in the methods of the present invention, are encompassed. This includes, in particular, microinjection, transfection, transduction, vesicle fusion, and electroporation.

[0093] Recombinant viruses have been widely applied to deliver genes to mammalian cells, especially stem cells. Viral vectors may be used in the context of the present invention.

[0094] Preferably, the viral vector for genetic modification of primary cells described herein is a retroviral vector, particularly a gammaretroviral vector. Gammaretrovirus (sometimes called mammalian C-type retrovirus) is a sister genus of the lentivirus clade and a member of the Orthoretrovirinae subfamily of the retrovirus family. Murine leukemia virus (MLV or MuLV), feline leukemia virus (FeLV), xenotropic murine leukemia virus-related virus (XMRV), and gibbon ape leukemia virus (GALV) are members of the gammaretroviral genus. Those skilled in the art are familiar with the techniques required for the use of gammaretroviruses in genetic modification of MSCs. For example, the vector described in Maetzig et al. (Gammaretroviral vectors: biology, technology and application, 2001, Viruses Jun;3(6):677-713) or a similar vector can be used. For example, the murine leukemia virus (MLV), a simple gammaretrovirus, can be transformed into an efficient vehicle for gene therapy by generating gammaretroviral-modified MSCs, delivering them to a subject, and then expressing a therapeutic transgene from the MSCs.

[0095] Lentiviruses are members of the Retroviridae family of viruses (M. Scherr et al., Gene transfer into hematopoietic stem cells using lentiviral vectors. CurrGene Ther. 2002 Feb; 2(1):45-55).

[0096] Lentiviral vectors are generated by deletion of all viral sequences except for the long terminal repeats and cis-acting packaging signals. The resulting vectors have a cloning capacity of approximately 8 kb. One of the distinguishing features of these vectors, as well as retroviral vectors, is their ability to transform dividing, non-dividing, and terminally differentiated cells.

[0097] The present invention further encompasses the administration of an expression vector to a subject in need thereof. A "vector" is any means for transferring a nucleic acid into a host cell. A preferred vector relates to a replicon to which another DNA segment can be attached to cause replication of the attached segment. As used herein, the term "vector" specifically refers to a means for introducing a nucleic acid into a cell in vitro, ex vivo, or in vivo. Viral vectors include, but are not limited to, retrovirus, adeno-associated virus, chickenpox, baculovirus, vaccinia, herpes simplex, Epstein-Barr, and adenovirus vectors.

[0098] Adenoviruses, or RNA viruses such as lentiviruses, or other retroviruses may be applied.

[0099] Adenoviruses have been used to generate a series of vectors for gene transfer and cell engineering. The initial generation of adenovirus vectors was produced by deleting the El gene (required for viral replication), generating vectors with a cloning capacity of 4 kb. Further deletion of E3 (involved in the host immune response) allowed for a cloning capacity of 8 kb. Further generations have been produced, including deletion of E2 and / or E4.

[0100] Non-integrating viral systems, such as adeno-associated viral vectors (AAV), represent a preferred embodiment of the gene therapy approach described herein due to their many advantages (see Asokan et al., Molecular Therapy, vol. 20, no. 4, 699-708). For example, AAV is particularly interesting for gene therapy because of its very limited ability to elicit an immune response in humans, a factor that positively impacts vector transduction efficiency while reducing the risk of immune-related pathologies. The AAV genome typically consists of approximately 4.7 kilobases of positive- or negative-sense single-stranded deoxyribonucleic acid (ssDNA) in length. The AAV genome consists of inverted terminal repeats (ITRs) at both ends of the DNA strand and two open reading frames (ORFs), the rep and cap sequences. The development of AAV as a gene therapy vector eliminated the vector's integrative ability by removing the rep and cap sequences from the vector's DNA. Any given desired gene and a promoter for driving the gene's transcription (e.g., TGIF2 of the present invention, as described herein) are inserted between inverted terminal repeats (ITRs), which aid in concatemer formation in the nucleus after the single-stranded vector DNA is converted into double-stranded DNA by the host cell's DNA polymerase complex. AAV-based gene therapy vectors typically form episomal concatemers in the host cell nucleus. In non-dividing cells, these concatemers remain intact throughout the life of the host cell. In dividing cells, the episomal DNA is not replicated along with the host cell's DNA, so the AAV DNA is lost during cell division.

[0101] With regard to viruses, these are preferably previously purified (e.g., by centrifugation on a cesium chloride gradient, column chromatography, plaque purification, etc.). They are typically present at concentrations of 10 per ml. 4 From 10 15 particles, preferably 10 5 From 10 12 It can be packaged in a proportion of particles.

[0102] Non-viral methods can also be used, including alternative strategies involving traditional plasmid transfer and targeted gene integration using integrase or transposase technology. These represent approaches to vector transformation that have the advantage of being efficient and, in many cases, site-specific in their integration. Physical methods for introducing vectors into cells are known to those skilled in the art. One example involves electroporation, which relies on the use of brief, high-voltage electrical pulses to overcome membrane capacitance and create transient pores in the membrane. One advantage of this method is that it can be used for both stable and transient gene expression in most cell types. Alternative methods involve the use of liposomes or protein transduction domains. Suitable methods are known to those skilled in the art and are not intended to limit embodiments of the present invention. Furthermore, delivery of RNA molecules, such as mRNA transfection, is included in the methods of the present invention for providing transcription factors from exogenous nucleic acids.

[0103] Furthermore, delivery of exogenous nucleic acid molecules for factor delivery can be achieved using transposable elements. For example, Sleeping Beauty, Tol2, and / or PiggyBac transposon systems or similar systems may be used. PiggyBac (PB) transposons are mobile genetic elements that efficiently transpose between vectors and chromosomes via a "cut-and-paste" mechanism. During transposition, the PB transposase recognizes transposon-specific inverted terminal repeats (ITRs) at both ends of the transposon vector, efficiently moving its contents from its original site and integrating them into the TTAA chromosomal site. The powerful activity of the PiggyBac transposon system allows for easy mobilization of a gene of interest between the two ITRs in the PB vector into the target genome. The TTAA-specific transposon PiggyBac is a highly useful transposon for the genetic engineering of various cells, including mammalian and human cells, particularly stem cells and iPSCs.

[0104] The supply of transcription factors and other factors used in the methods of the present invention can be transient or permanent. For example, when supply is achieved by expression from a nucleic acid molecule, the expression of the transcription factor can be permanently active under the control of a constitutive promoter or a promoter that is active in initial cells and induced photoreceptor cells. Alternatively, the expression and supply of the transcription factor can be transient. This is because the nucleic acid molecule encoding the transcription factor is removed or eliminated from the cell, or because expression is controllable and can be turned on and off, for example, by using controlled transcriptional activation. In the context of the present invention, transient expression refers to only the temporary expression of a factor derived from a nucleic acid molecule, as opposed to permanent expression. Transient expression can be based on expression from a delivered mRNA molecule, which is degraded over time in the cell, so expression only occurs if the delivered mRNA is not degraded.

[0105] In another example, transient expression can occur through the induction of gene expression from an exogenous DNA molecule containing a controllable genetic element that drives the expression of the encoded gene, thus including inducible gene expression. In such a system, gene expression can be controlled externally, for example, through the administration of a compound, such as an antibiotic molecule or a drug, that activates gene expression. Such systems have been fully described in the art and are known to those skilled in the art.

[0106] The gene expression systems that can be used in the present invention are systems specifically designed for the production of a selected gene product. This is usually a protein, but can also be RNA, such as microRNA. Expression systems are typically composed of a gene encoded by DNA and the molecular machinery required to transcribe the DNA into mRNA and translate the mRNA into protein using provided reagents. Thus, expression systems are often artificial in some ways, although certain parts of the machinery required for gene expression may be provided by the target cell.

[0107] For example, inducible and / or controlled gene expression can be achieved using tetracycline-controlled transcriptional activation. Tetracycline-controlled transcriptional activation is a method of inducible gene expression in which transcription is reversibly turned on or off in the presence of the antibiotic tetracycline or one of its derivatives (e.g., doxycycline). Tetracycline-controlled gene expression is based on the mechanism of resistance to tetracycline antibiotic treatment found in Gram-negative bacteria. The Ptet promoter expresses the repressor TetR and the protein TetA, which expels tetracycline antibiotics from bacterial cells. The difference between the Tet-On and Tet-Off systems is not whether a transactivator turns genes on or off, but rather that both proteins activate expression. The difference relates to their respective responses to tetracycline or doxycycline (Dox, a more stable tetracycline analog). Tet-Off activates expression in the absence of Dox, while Tet-On activates in the presence of Dox.

[0108] In the context of this invention, the term transcription factor (TF) refers to a protein that controls the rate of transcription of genetic information from DNA to messenger RNA by binding to specific DNA sequences. The function of a transcription factor is to regulate (turn on and off) genes so that they are expressed in the right amount at the right time in the right cells throughout the life of a cell and an organism. Groups of transcription factors work in concert to direct cell differentiation, cell division, cell proliferation, and cell death throughout life, cell migration and organization (body plan) during embryonic development, and intermittently in response to extracellular signals such as hormones. Transcription factors function alone or in complexes with other proteins by promoting (as activators) or blocking (as repressors) the recruitment of RNA polymerase (the enzyme that transcribes genetic information from DNA to RNA) to specific genes. A defining characteristic of transcription factors is that they contain at least one DNA-binding domain (DBD), which binds to specific DNA sequences adjacent to the genes they control.

[0109] Transcription factors can be used for reprogramming or directed differentiation of mammalian cells into different cell types. Induction of different cell types in initial / starting cells can be achieved by providing one or more transcription factors. In the context of the present invention, the term "initial cell" refers to a cell used as a starting point for inducing a photoreceptor phenotype in this cell, in which at least the transcription factor GON4L is provided in the cell. In the context of the present invention, any type of cell, preferably a mammalian cell, can be used as the initial cell. Preferably, the initial cell is a human cell. Cells are the basic structural, functional, and biological units of all known organisms. Cells are the smallest units of life. Cells are often referred to as the "building blocks of life."

[0110] Preferred initial cells of the present invention are pluripotent or multipotent mammalian cells, including stem cells. Preferably, the initial cells are mammalian, preferably human, cells, including human induced pluripotent stem cells (iPSCs). iPSCs are a type of pluripotent stem cell that can be generated directly from adult cells. iPSCs can not only grow indefinitely in cell culture, but also give rise to all other cell types in the body or in individual mammalian organisms, e.g., neurons, cardiac cells, pancreatic cells, and liver cells, and represent a single source of cells that can be used to replace cells lost due to injury or disease. The most well-known type of pluripotent stem cell is the embryonic stem cell. However, because their generation involves the manipulation of preimplantation embryos, much ethical controversy has surrounded their use. Furthermore, because embryonic stem cells can only be obtained from embryos, it has thus far been impossible to create patient-specific embryonic stem cell lines. Because iPSCs can be derived directly from adult tissues, they not only avoid the need for embryos but can also be created in a patient-specific manner, meaning that each individual can have their own unique pluripotent stem cell line. This unlimited supply of autologous cells can be used to generate grafts without the risk of immune rejection. Furthermore, iPSCs and iPSC-derived cells can be used for personalized drug discovery and understanding the basis of patient-specific diseases. This also applies to the induced photoreceptor cells of the present invention, which can be derived from human patient-specific iPSCs. iPSCs are typically obtained by introducing the products of a specific set of pluripotency-associated genes, or "reprogramming factors," into a given cell type. The original set of reprogramming factors is the transcription factors Oct4 (Pou5f1), Sox2, cMyc, and Klf4. While this combination is most common in iPSC production, each factor can be functionally replaced with related transcription factors, miRNAs, small molecules, or even unrelated genes such as lineage specifiers. Such replacement of factors required for cellular reprogramming also applies to other cellular reprogramming efforts.

[0111] Furthermore, the early cells used in connection with the present invention are not photoreceptors in the sense of the present invention, but are fibroblasts, retinal progenitor cells (RPCs), retinal pigment epithelial (RPE) cells, Muller glial cells, and other cell types found in the eye and retina.

[0112] The method of the present invention involves supplying the transcription factor GON4L to early cells. GON4L is a protein encoded by the GON4L gene in humans. It is a nuclear protein containing two serine phosphate sites and a lysine-glutamine bridge and is thought to be a transcription factor. GON4L homologs play conserved roles in cell cycle regulation and / or embryonic patterning in plants, nematodes, flies, mice, and fish. However, the contribution of GON4L or other chromatin factors to morphogenesis is not particularly well understood.

[0113] Preferably, the present invention further relates to the provision of one or more transcription factors selected from CRX, NEUROD1, NR2E1, NR2E3, NRL1, OTX2, ONECUT1, PAX6, RAX, RORB, RXRG, SIX3, SIX6, SOX2, THRB and VSX2, which have been described as being highly relevant to the differentiation and development of photoreceptor cells.

[0114] OTX2 is a protein encoded by the OTX2 gene in humans. This gene encodes a member of the bicoid subfamily of homeodomain-containing transcription factors. This encoded protein functions as a transcription factor and may play a role in brain and sensory organ development. A similar protein in mice is required for proper forebrain development. Two transcript variants encoding distinct isoforms of this gene have been identified. Other alternative splicing variants may exist, but their full-length sequences have not been determined.

[0115] NEUROD1 / NeuroD1 (neurogenic differentiation 1), also known as b2, is a NeuroD-type transcription factor. It is encoded by the human gene NEUROD1. It is a member of the NeuroD family of basic helix-loop-helix (bHLH) transcription factors. This protein forms heterodimers with other bHLH proteins and activates the transcription of genes containing specific DNA sequences known as E-boxes. It regulates the expression of the insulin gene, and mutations in this gene cause type II diabetes. NeuroD1 has been shown to convert reactive glial cells into functional neurons in vivo in the mouse brain.

[0116] In the context of the present invention, one or more transcription factors may be provided at the protein level or in the form of a nucleic acid encoding the transcription factor.

[0117] Preferred amino acid sequences of GON4L, NEUROD1 and OTX2 are listed in Table 1.

[0118] Table 1: Amino acid sequences of preferred transcription factors of the present invention [Table 1] JPEG0007743061000002.jpg195141JPEG0007743061000003.jpg195141JPEG0007743061000004.jpg128141

[0119] In the context of the present invention, the provision of GON4L isoform B as set forth in SEQ ID NO: 3 and / or OTX2 isoform as set forth in SEQ ID NO: 6 is particularly advantageous.

[0120] The present invention also relates to functionally similar sequences of each transcription factor. Protein modifications to the transcription factors of the present invention, which may occur through amino acid sequence substitution, and nucleic acid sequences encoding such molecules, are also within the scope of the present invention. Substitutions, as defined herein, are modifications made to the amino acid sequence of a protein, whereby one or more amino acids are replaced with the same number of (different) amino acids, producing a protein containing an amino acid sequence different from that of the original protein. In some embodiments, this modification does not significantly alter the function of the protein. Like additions, substitutions can be natural or artificial. It is well known in the art that amino acid substitutions can be made without significantly altering the function of a protein. This is particularly true when the modification involves "conservative" amino acid substitutions, which involve replacing one amino acid with another amino acid of similar properties. Such "conservative" amino acids can be natural or synthetic amino acids that, due to their size, charge, polarity, and structure, can be substituted without significantly affecting the structure and function of the protein. In many cases, many amino acids can be substituted with conservative amino acids without adversely affecting the function of the protein.

[0121] Generally, the following conserved amino acid groups are represented: nonpolar amino acids Gly, Ala, Val, Ile, and Leu; nonpolar aromatic amino acids Phe, Trp, and Tyr; neutral polar amino acids Ser, Thr, Cys, Gin, Asn, and Met; positively charged amino acids Lys, Arg, and His; and negatively charged amino acids Asp and Glu. This list is not exhaustive. For example, it is well known that Ala, Gly, Ser, and in some cases Cys can substitute for each other, even though they belong to different groups.

[0122] As explained herein, in the context of the present invention, one or more transcription factors may be provided at the protein level or in the form of a nucleic acid encoding the transcription factor.

[0123] Nucleic acid sequences of the present invention include nucleic acid sequences encoding the GON4L, NEUROD1 and OTX2 protein sequences and functionally similar sequences shown in Table 1. Preferred nucleic acid sequences encoding the GON4L, NEUROD1 and OTX2 protein sequences are listed in Table 2.

[0124] The transcription factor of the present invention can comprise a protein tag, for example, by using an antibody against the protein tag, allowing the transcription factor supplied into cells to be easily identified through standard techniques.The preferred protein tag that can be encoded by the nucleic acid sequence of the present invention is V5 tag.Alternative tags can be used instead of V5 tag.Such alternative tags are well known in the art and can be selected by those skilled in the art.

[0125] Table 2: Nucleic acid sequences of preferred transcription factors of the present invention [Table 2] JPEG0007743061000006.jpg194141JPEG0007743061000007.jpg194141JPEG0007743061000008.jpg19414 1JPEG0007743061000009.jpg194141JPEG0007743061000010.jpg194141JPEG0007743061000011.jpg39139

[0126] In another embodiment, the present invention encompasses the use of one or more nucleic acid molecules encoding one or more transcription factors, in particular GON4L and optionally NEUROD1 and OTX2, selected from the group comprising: a) one or more nucleic acid molecules comprising a nucleotide sequence encoding human GON4L, preferably human GON4L as set forth in SEQ ID NO: 8, and optionally a nucleotide sequence encoding NEUROD1, preferably NEUROD1 as set forth in SEQ ID NO: 10, and OTX2, preferably OTX2 as set forth in SEQ ID NO: 12; b) one or more nucleic acid molecules that are complementary to the nucleotide sequence shown in a); c) one or more nucleic acid molecules that undergo hybridization under stringent conditions with the nucleotide sequence shown in a) or b); d) one or more nucleic acid molecules comprising a nucleotide sequence having sufficient sequence identity to be functionally similar to the nucleotide sequence set forth in a), b), or c); e) one or more nucleic acid molecules that, as a result of the genetic code, are degenerate to the nucleotide sequences shown in a) through d); and f) One or more nucleic acid molecules set forth in the nucleotide sequences of a) to e) that are modified by deletions, additions, substitutions, translocations, inversions and / or insertions and / or are functionally similar to the nucleotide sequences set forth in a) to e).

[0127] That is, the present invention encompasses nucleic acid molecules having at least 60%, preferably 70%, more preferably 80%, and particularly preferably 90% sequence identity with nucleic acid molecules encoding GON4L, and preferably NEUROD1 and OTX2.

[0128] Sequence variants of the claimed nucleic acids and / or proteins that maintain the above-described properties of the invention, e.g., as defined by the provided % sequence identity, are also included within the scope of the present invention. Such variants represent alternative sequences, but maintain essentially the same properties, such as GON4L function and, optionally, NEUROD1 and OTX2 function, since the specific sequences provided are known as functional analogs or functional analogs. Sequence identity relates to the percentage of identical nucleotides or amino acids when performing sequence alignments using software such as BLAST.

[0129] It will be understood by those skilled in the art that, due to the degeneracy of the genetic code, there are many nucleotide sequences that encode the polypeptides described herein. Some of these polynucleotides have minimal homology or sequence identity with the nucleotide sequence of any native gene. Nevertheless, polynucleotides that vary due to differences in codon usage are specifically anticipated by the present invention. Deletions, substitutions and other changes in the sequences that fall within the described sequence identity are also included in the present invention.

[0130] In the context of the present invention, the term "microRNA" or microRNA / miRNA refers to small, non-coding RNA molecules found in plants, animals, and some viruses that function in RNA silencing and post-transcriptional regulation of gene expression. miRNAs function through base pairing with complementary sequences within mRNA molecules. As a result, these mRNA molecules are silenced by one or more of the following processes: (1) cleavage of the mRNA strand into two parts, (2) destabilization of the mRNA by shortening its poly(A) tail, and (3) reduced efficiency of translation of the mRNA into protein by ribosomes. miRNAs are abundant in many mammalian cell types and are believed to target approximately 60% of genes in humans and other mammals. In the context of the present invention, the provision of human miR-182 (Gene ID: 406958) and miR-183 (Gene ID: 406959) may be particularly advantageous.

[0131] The term cell cycle inhibitor refers to any kind of molecule, such as a small chemical molecule, but also to proteins, nucleic acids, or other molecules that slow or stop cell cycle progression through various mechanisms. Cell cycle arrest can be induced at various stages and reduces the rate of cell division and the number of cells actively progressing through the cell cycle.

[0132] In the context of the present invention, the use of the cell cycle inhibitor AraC is particularly preferred. AraC, also known as cytarabine or cytosine arabinoside, is used as a chemotherapy drug to treat acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), chronic myeloid leukemia (CML), and non-Hodgkin's lymphoma. Many cell cycle inhibitors are known in the art and can be identified by one of skill in the art, including, but not limited to, pladienolide B, methotrexate, roscovitine, daidzein, baicalein, indirubin-3'-oxime, epothilone B, narciclasine, AZD 5438, ABT751, YC 1, 10058-F4, 8-chlorodenosine, DIM, plumbagin, pyridostatin pentahydrochloride, SKPin C1, CPI203, CGP 60474, XL 413 hydrochloride, CHMFL-FLT3-122, a potent and selective FLT3 inhibitor, WYE 687 dihydrochloride, NSC 23005 sodium.

[0133] Administration of a cell cycle inhibitor refers to the addition of a molecule to the cell culture medium, where the molecule is thus made available to the cells. The term administration also includes all types of supply of factors described herein in the sense of making the factor available in the treated cells, such as the initial cells of the invention.

[0134] Thus, the factor supplied may be a cell cycle inhibitor.

[0135] In an embodiment of the method of the present invention, the initial cells are cultured on a basement membrane-like matrix, such as Matrigel, or another gelatinous protein mixture, such as specific collagen or laminin molecules that support the development or maintenance of photoreceptor cells.

[0136] Matrigel: A gelatinous protein mixture secreted by Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells. Matrigel resembles the complex extracellular environment found in many tissues and is used by cell biologists as a substrate (basement membrane matrix) for culturing cells. Similarly, in the present invention, it is possible to provide a different gelatinous protein mixture in a specific cell culture system that provides a favorable microenvironment for cultured cells, an environment that promotes the differentiation of early cells toward a photoreceptor-like phenotype. This can be achieved by providing a matrix containing specific laminins or other extracellular matrix proteins that are abundant in the extracellular matrix of the retina. In particular, matrices for culturing the cells of the present invention and practicing the methods of the present invention can include poly-L-ornithine, poly-L-lysine, poly-D-lysine, and / or laminin (In), preferably laminins with β-2 chains such as In323, In423, In523, and / or In521.

[0137] In the context of the present invention, the term "photoreceptor reporter system" refers to any type of system that can be used to determine the development of a photoreceptor-like phenotype, indicative of the differentiation of early cells into photoreceptor cells or their precursor cells. Such systems typically use exogenous nucleic acid sequences encoding a reporter or marker gene. Such reporter genes can preferably encode fluorescent proteins and can be easily detected upon expression by standard techniques, such as microscopy, cytometry, and others. Expression of such reporter or marker genes can be under the control of a genetic element, such as a promoter sequence of a gene typically expressed in photoreceptor cells or their precursor cells, or a portion of such a sequence. Examples of photoreceptor-specific genes whose genetic control elements can be used in connection with a photoreceptor reporter system include genes encoding cone arrestin, rhodopsin, recoverin, NCAM, OTX, CRX, RCVRN, RHO, OPN1 SW, OPN1 MW, and / or OPN1 LW. One skilled in the art can further identify suitable promoter sequences by identifying photoreceptor-specific genes or combinations of such genes by examining typical gene expression profiles of photoreceptor cells available in the art. The design of cell-type-specific reporter systems is a well-defined technique known to those skilled in the art. Marker genes can also encode proteins that confer resistance to compounds such as antibiotics, allowing for the selection of cells from a mixed culture system that express such markers under the control of a photoreceptor-specific promoter sequence in the presence of the compound, while other cells cannot survive in the presence of the respective compound.

[0138] Another method for identifying induced photoreceptor cells in a mixed culture containing initial cells may be to detect the loss of markers of the initial cells, such as the loss of Tra1-60 expression in the case of iPSCs as initial cells. Cells can be characterized and induced photoreceptor cells can be identified and isolated by flow cytometry using the expression of fluorescent marker proteins and / or surface protein expression patterns typical of photoreceptor cells and their progenitors, compared to the surface marker patterns of the initial cells.

[0139] In the context of the present invention, the term retinopathy refers to damage to the retina of the eye that can lead to vision loss. Often, retinopathy refers to retinal vascular disease or damage to the retina caused by abnormal blood flow. Age-related macular degeneration is included under the general term retinopathies. Retinopathy includes retinal vascular disease and can be broadly classified into proliferative and non-proliferative forms. In many cases, retinopathy is an ocular manifestation of a systemic disease, such as that seen in diabetes or hypertension.

[0140] Retinopathy is further associated with macular degeneration, also known as age-related macular degeneration (AMD or ARMD), a condition that can cause blurred or no vision in the center of the visual field. Over time, patients may experience a gradual deterioration of vision that can affect one or both eyes. While not resulting in complete blindness, loss of central vision can make it difficult to recognize faces, drive, read, or perform other activities of daily living. Visual hallucinations may also occur, but these do not represent a psychiatric disorder. Macular degeneration typically occurs in older adults, but genetic factors and smoking also play a role.

[0141] It is thought to be caused by damage to the macula of the retina. The severity is divided into early, intermediate, and late types, all of which can be treated by using the cells of the present invention. The late type is further divided into "dry" and "wet" types, with the dry type accounting for 90% of cases, and all types can be treated by transplanting the cells of the present invention.

[0142] Retinal degeneration is a type of retinal disease characterized by the deterioration of the retina due to the progressive death of its cells. Retinal degeneration can occur for several reasons, including arterial or venous blockage, diabetic retinopathy, RLF / ROP (retrolental fibroplasia / retinopathy of prematurity), or disease (usually hereditary), and can manifest in many different ways, including visual impairment, night blindness, retinal detachment, light sensitivity, tunnel vision, and loss of peripheral vision to complete loss of vision. Among retinal degenerative diseases, retinitis pigmentosa (RP) is a significant example. Human hereditary retinal degenerative diseases exhibit genetic and phenotypic heterogeneity in underlying causes and clinical outcomes. There are various possible causes of retinal degeneration, including disruption of genes involved in phototransduction, rhodopsin molecule biosynthesis and folding, and structural support of the retina. Mutations in the rhodopsin gene account for approximately 25% to 30% of all cases of autosomal dominant retinitis pigmentosa (adRP) in North America. There are many mechanisms of retinal degeneration resulting from rhodopsin mutations or mutations that involve or affect rhodopsin function. One mechanism of retinal degeneration is overexpression of rhodopsin. Another mechanism, in which mutations cause rhodopsin truncation, has been found to affect rod function and increase the rate of photoreceptor degeneration.

[0143] Cell transplantation is a novel therapeutic strategy to restore visual responses to degenerated adult neural retina, and transplanted postmitotic photoreceptor precursors have been shown to be able to functionally integrate into the adult mouse neural retina. [Example]

[0144] The present invention is further illustrated by the following examples, which are not intended to limit the scope of the invention but rather represent preferred embodiments or aspects of the invention provided for further illustration.

[0145] Although it is possible to obtain photoreceptors by direct reprogramming from small numbers of fibroblasts, an efficient 2D protocol for generating photoreceptors in vitro from human induced pluripotent stem cells (hiPSCs) needs to be established. Forward programming relies on the ability of transcription factors (TFs) to activate different differentiation pathways in stem cells. We performed a library screen of a transcription factor library with the aim of finding combinations of transcription factors that promote efficient differentiation of stem cells into photoreceptors.

[0146] method

[0147] General Procedure

[0148] A transcription factor library consisting of 1,748 human transcription factors was used to generate specific retinal cell types (rod and cone photoreceptors). Photoreceptor-specific reporter constructs were used that were activated during specific states of photoreceptor development (e.g., retinal and preneural fold homeobox (RX), cone rod homeobox (CRX), cone arrestin-3 (CAR), and rhodopsin (RHO)) and drive the expression of fluorescent proteins and selectable markers from different ubiquitous promoters. In some cases, multiple reporter cassettes were integrated into a single iPS cell line via lentiviral gene transfer. Further reporter cell lines were generated by introducing reporter cassettes using the PiggyBac system. Corresponding knock-in cell lines were also generated. These reporter human iPS cell lines were then tested for expression in retinal organoids, and the best-performing cell lines were selected for application of the transcription factor library. Upon transcription factor induction, we screened fluorescently labeled photoreceptors (CAR and RHO) and / or their progenitors (RX and CRX) (Figure 4, right). In parallel, we used existing knowledge of transcription factors acting during photoreceptor development and specifically applied these selected transcription factors in a biased approach (Figure 4, left). In the latter experiment, we induced RX, SIX3, SIX6, LHX2, TLL, OTX2, PAX6, SOX1, SOX2, CRX, ONECUT1, VSX2, NRL, TRB2, NEUROD1, NR2E3, RXRG, and RORB (8). These were PCR amplified from library pools and applied individually or in combination. We also combined the two library approaches. To minimize the risk of failure and identify specific developmental stages, we generated transcriptomic data from both approaches. We compared the transcriptomic profiles and genetic programs leading to photoreceptors. To identify and debug key steps and pitfalls in stem cell-derived photoreceptor generation, profiling intermediately arrested photoreceptor progenitors is of particular interest.

[0149] Rod and cone photoreceptors can be easily distinguished by their specific gene expression profiles. Generally, these cell types are well characterized in vivo, allowing comparative troubleshooting. Cell characterization involves applying specific antibodies against phototransduction cascade members and functional patch-clamp recording to characterize induced photoreceptors. We previously showed that upregulation of two microRNAs (miR-182 and miR-183) in stem cell-derived retinal photoreceptors is sufficient to promote the formation of the light-sensitive compartment (outer segment) (11). Therefore, overexpression of these non-coding RNAs is beneficial for the functional maturation of photoreceptors.

[0150] Specific Experiments

[0151] Reporter hiPSC lines were transduced with a lentiviral library of 16 known transcription factors, followed by a comprehensive library of 1,748 human transcription factors. HiPSCs lacking transcription factors were killed by selection using the marker included in the lentiviral cassette. A portion of the cells was used for transcription factor induction by treatment with doxycycline (dox) for 10 days. Of the transduced and induced cells, 87 were fluorescently labeled and sorted into individual wells (Figure 5). Single-cell RNA was extracted and split for single-cell qPCR analysis and detection of overexpressed transcription factors. In particular, OTX, CRX, RCVRN, RHO, OPN1 SW, and OPN1 LW were identified using RT primers specific for the overexpressed transcription factors. Transcription factor detection was performed by amplifying the transcription factors from cDNA by PCR, loading them onto a gel, and excising and sequencing the amplified DNA bands. Based on the identification of overexpressed transcription factors, preferred transcription factors of the present invention, specifically GON4L, NEUROD1, and OTX2, were identified as being particularly efficient at inducing the cone phenotype (Figure 6). The transcription factor combination was validated in hiPSC reporter lines using flow cytometry to detect loss of a pluripotency marker (Tra1-60) and upregulation of a neuronal marker (NCAM), as well as fluorescence from the reporter cassette.

[0152] Nucleic acid sequences encoding the transcription factors used

[0153] The nucleic acid sequences encoding the transcription factors GON4L, NEUROD1 and OTX2 used in the experiments of the present invention are shown in SEQ ID NO: 9, SEQ ID NO: 11 and SEQ ID NO: 13, as listed in Table 2. Note that all three transcription factors have a V5 tag at their 3' end.

[0154] cell culture

[0155] PGP1 (GM23338, Coriell), ATCC DYS0100 (ATCC® ACS-1019™, ATCC), and CRTD5 (CRTD reprogrammed in the iPSC facility; Kutsche et al. Cell Systems 2018, Oct 24;7(4):438-452) human induced pluripotent stem cells (hiPSCs) were cultured in mTeSRI medium (05850, StemCell Technologies). Prior to adding hiPSCs, standard tissue culture well plates were coated with hESC-qualified Matrigel matrix (354277, Corning) and incubated for 60 minutes at room temperature. hiPSCs were cultured under standard conditions (5% CO2, 37°C), with daily changes of mTeSRI medium. For passaging, hiPSCs were detached from the wells by adding TrypLE Express (12604013, Thermo Fisher Scientific), washed with phosphate-buffered saline (PBS, pH 7.2; 14190169, Thermo Fisher Scientific), spun down at 400 x g, and added to fresh Matrigel-coated tissue culture wells in mTeSRI medium containing 3 pg / ml InSolution Y-27632 rho kinase inhibitor (688001, Merck Millipore). Alternatively, cells were frozen in mFreSR medium (05854, StemCell Technologies).

[0156] Stable integration of inducible transcription factors or photoreceptor reporter cassettes was achieved using the PiggyBac transposon system. All vector elements between the 5' core insulator and the SV40 polyA site of the PiggyBac vector backbone PB-TRE-dCas9-VPR13 (Addgene plasmid #63800; Chavez et al., 2015, Nat Methods. 2015 Mar 2. doi:10.1038 / nmeth.3312) were replaced with the corresponding DNA fragments. Ten pg of the plasmid was mixed with 2 pg of the Super PiggyBac transposase expression vector (PB210PA-1-S, Biocat) and electroporated into hiPSCs using Lonza 4D X units, pulse CB-156, and the P3 Primary Cell 4D-NucleofectorKit L (V4XP-3024, Lonza). According to the selection cassette chosen, blasticidin (25 pg / ml), puromycin (0.5-1 pg / ml), or hygromycin B (250 pL / ml) was applied.

[0157] Standard lentiviral transduction was performed for transcription factor screening. Cell numbers and viral particles were adjusted to achieve a multiplicity of infection of 1. PGP1 iPSCs containing a photoreceptor reporter cassette were serially transduced with either an unbiased transcription factor library (1748 transcription factors each contained in the lentiviral pLIX_403 backbone (Addgene plasmid 41395)) or a library of selected transcription factors (backbone derived from Addgene plasmid 61473) and then selected by the corresponding selectable markers.

[0158] Transcription factor induction for differentiating hiPSCs: Transcription from the TeTOn promoter was induced by applying 0.5 pg / ml doxycycline (D9891, Sigma-Aldrich) to mTeSRI medium.

[0159] Photoreceptor reporter system details

[0160] The photoreceptor reporter system is based on the PiggyBac vector PB-TRE-dCas9-VPR13 (Addgene plasmid #63800; Chavez et al., 2015, Nat Methods. 2015 Mar 2. doi:10.1038 / nmeth.3312). All vector elements between the 5' core insulator and SV40 polyA site were replaced with either an eGFP cassette driven from the mouse cone arrestin promoter (mCAR, Busskamp et al. Science 2010 Jul 23;329(5990):413-7) or the human rhodopsin promoter (RHO, Busskamp et al. Science 2010 Jul 23;329(5990):413-7) driving the red fluorescent protein dsRED. Downstream of the fluorescent protein, a woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) and a blasticin selection cassette driven by the ubiquitin C promoter (both obtained from Addgene plasmid 61473) were added. The corresponding vectors, pb-mCAR-EGFP-UBC-Blasti and pb-Rho-dsRed-UBC-Blasti, were co-nucleofected into PGP1 hiPSCs, and transgenic clones harboring both constructs were selected.

[0161] Concentrations of cell cycle inhibitors and other components used in each experiment

[0162] Cytosine bD-arabinofuranoside hydrochloride (Ara-C, C6645, Sigma) was used at a final concentration of 5 mM for 24 h to deplete dividing cells in neuronal cultures.

[0163] result

[0164] Eighty-seven percent of sorted cells were qPCR-positive for at least one of the photoreceptor-specific genes tested, demonstrating the cell-type accuracy of the screen. Several of the tested transcription factor combinations, including GON4L and, in some cases, OTX2 and NEUROD1, resulted in a significant loss of the pluripotency marker Tra1-60 and an upregulation of the neuronal marker NCAM after 5 days of overexpression (hiPSCs: 0.47 ± 0.07%, hiPSC-TFs: 75.23 ± 3.7%; mean ± SEM, Welch's two-tailed t-test; p = 0.002), indicating that cells were differentiating toward a neuronal lineage.

[0165] Furthermore, fluorescence microscopy and flow cytometry detected GFP-positive cells after 10 days, suggesting the presence of cone photoreceptors.

[0166] conclusion

[0167] We systematically screened transcription factors based on in vivo studies and a human transcription factor library to find combinations that would help us achieve our ultimate goal of engineering human photoreceptors in vitro. Our data suggest that known factors are insufficient to promote photoreceptor differentiation and demonstrate that generating photoreceptors from hiPSCs requires additional transcription factors, particularly GON4L. The combination of GON4L with OTX2 and NEUROD1 was particularly advantageous for efficiently inducing photoreceptor differentiation. Because in vitro engineered photoreceptors can be generated in sufficient quantities within 10 days, compared with hundreds of days when dissociated from 3D human retinal organoids, they may serve as donor material for cell transplantation to treat blindness.

[0168] Transplantation of induced human photoreceptors into the retina of blind mice

[0169] As mentioned previously, there are many approaches to transplant photoreceptors into mouse models of retinal degeneration. For this purpose, mouse photoreceptor progenitor cells can be harvested and injected into the subretinal space of blind retinas (12-14). Furthermore, rod photoreceptor progenitor cells derived from 3D organoids can be isolated and successfully transplanted (15). A small fraction of these mouse cells has been shown to functionally integrate into the host mouse retina. Because induced human photoreceptors have not been used previously, we are the first to use these cells as starting material for transplantation into the retinas of blind mice.

[0170] To visualize and functionally test the transplanted photoreceptors, we plan to tag these cells prior to injection with a fluorescent reporter fused to a hyperpolarizing optogenetic tool (16, 17). In addition to fluorescence detection, we trigger light sensitivity by stimulating the optogenetic tool with light and then record the light response. Because endogenous photoreceptors in disease mouse models are insensitive to light, all light responses can be traced back to the transplanted, and therefore functionally integrated, cells. The intrinsic phototransduction cascade of rods and cones is logarithmically more sensitive than optogenetic sensors. Therefore, controlling the light level for stimulation allows for the distinction between endogenous and optogenetic light responses.

[0171] To measure the success of reactivation, we will perform patch clamp recordings directly from transplanted photoreceptors. To test whether cones are integrated into existing retinal circuits, we will record by patch clamp or MEA from retinal ganglion cells. Recovered vision will also be investigated using behavioral tests, as previously demonstrated (16). In addition to functional studies, we will perform immunohistochemical analysis at the CRTD light microscopy facility, followed by confocal and electron microscopy. We will also study the transcriptomic profiles of successfully integrated human photoreceptors and compare them with unsuccessful ones to determine the critical biological parameters for improving cone integration.

[0172] References 1. Ishii, T., Yin, C., Seko, Y., Umezawa,A. & Kaneda, M. Variation in the Phenotype of Photosensitive Cells Producedfrom Human Fibroblast Cell Lines. 1 Nippon Med Sch85, 1 10-1 16 (2018). 2. Seko, Y. et al. Derivation of human differentialphotoreceptor cells from adult human dermal fibroblasts by defined combinationsof CRX, RAX, OTX2 and NEUROD. Genes Cells 19, 198-208 (2014). 3. Gonzalez-Cordero, A. et al. Recapitulation of Human Retinal Development fromHuman Pluripotent Stern Cells Generates Transplantable Populations of ConePhotoreceptors. Stern Cell Reports 9, 820-837 (2017). 4. Slembrouck-Brec, A., Nanteau,C., Sahel, J.A., Goureau, 0. & Reichman.S. Defined Xeno-free andFeeder-free Culture Conditions for the Generation of Human IPSC- derivedRetina! Cell Models. 1 Vis Exp (2018). 5. Volkner, M., Kurth, T.& Karl, M.O. The Mouse Retinal Organoid Trisection Recipe: Efficient Generation of 3D Retinal Tissue from Mouse Embryonic Stern Cells. Methods Mol Biol 1834, 119-141 (2019). 6. Volkner, M. et al. Retinal Organoids fromPluripotent Stern Cells Efficiently Recapitulate Retinogenesis. Stern Cell Reports 6,525-538 (2016). 7. Lakowski, 1. et al. Isolation of HumanPhotoreceptor Precursors via a Cell Surface Marker Panel from SternCell-Derived Retina! Organoids and Fetal Retinae. Stern Cells 36, 709-722(2018). 8. Hennig, A.K., G.H. Peng, and S. Chen, Brain Res,2008 Feb 4;1 192:1 14-33. 9. Zuber, M.E., Curr Top Dev Biol,2010;93:29-60. 10. Zuber, M.E., et al., Development, 2003, Nov;130(21 ):5155-67. 1 1 . Busskamp, V., et al., Neuron, 2014, Aug6;83(3):586-600. 12. MacLaren, R.E., et al., Nature, 2006, Nov9;444(71 16):203-7. 13. Pearson, R.A., et al., Nature, 2012, May 3;485(7396):99-103. 14. Santos-Ferreira, T., et al., Stem Cells, 2015 Jan;33(1 ):79-90. 15. Gonzalez-Cordero, A., et al., Nat Biotechnol,2013 Aug;31 (8):741 -7. 16. Busskamp, ​​V., et al., Science, 2010, Jul23;329(5990):413-7. 17. Chuong, AS, et al., Nat Neurosci,2014, Aug;17(8):1 123-9.

[0173] Drawing Terminology cone Selected TFs Selected transcription factors TF Library Transcription factor library Lentiviral production Conditional PR reporter iPS FACS and TF identification Debugging End point characterization functional tests revealing in vitro PR neurogenesis pathways Transplanting human photoreceptors in blind mouse retinas cell cell qPCR photoreceptor gene panel Transcription factors Biased group Fold change days Seeding Daily media change Dissociating and FACS of GFP+ cells GFP+ viable GFP+survival GFP viable analysis

Claims

1. A method for producing photoreceptor cells induced in vitro from early cells, the method comprising providing transcription factors (TFs) including GON4L, OTX2 and NEUROD1 to early cells in vitro, wherein the early cells are pluripotent or multipotent mammalian cells, fibroblasts, retinal progenitor cells (RPCs), retinal pigment epithelial (RPE) cells, Muller glial cells, or other cells found in the eye or retina that are not photoreceptors.

2. The method of claim 1 , wherein the initial cells are induced pluripotent stem cells (iPSCs).

3. 3. The method of claim 1 or 2, further comprising supplying the initial cells with one or more transcription factors selected from CRX, NR2E1, NR2E3, NRL1, ONECUT1, PAX6, RAX, RORB, RXRG, SIX3, SIX6, SOX2, THRB and VSX2.

4. The method of any one of claims 1 to 3, wherein the transcription factors are expressed from one or more exogenous nucleic acid molecules in the initial cell.

5. The method of claim 4 , wherein the one or more exogenous nucleic acid molecules are viral vectors.

6. The method of any one of claims 1 to 5, wherein the initial cells are supplied with the transcription factors for at least 4 days.

7. The method of any one of claims 1 to 6, wherein the transcription factor is transiently expressed and / or expression is induced in the primary cell.

8. The method of any one of claims 1 to 7, comprising administering a cell cycle inhibitor to the early stage cells.

9. 9. The method of claim 8, wherein the cell cycle inhibitor is AraC and the cell cycle inhibitor is administered after providing the transcription factor.

10. 10. The method of any one of claims 1 to 9, wherein the induced photoreceptor cells produced from the initial cells are determined by a photoreceptor reporter system present in the initial cells, said reporter system comprising one or more photoreceptor-specific promoter sequences and one or more reporter genes and / or selectable markers.

11. The method of any one of claims 1 to 10, wherein the generation of induced photoreceptor cells is determined by the expression of endogenous recoverin, NCAM, OTX, CRX, RCVRN, RHO, OPN1 SW and / or OPN1 LW.

12. The method of any one of claims 1 to 11, wherein the induced photoreceptor cells are cones.

13. 13. An induced photoreceptor cell obtained by the method of any one of claims 1 to 12, said cell comprising one or more exogenous nucleic acid molecules encoding the transcription factors (TFs) GON4L, OTX2 and NEUROD1.

14. A kit for producing induced photoreceptor cells derived from initial cells according to the method of any one of claims 1 to 12, comprising: a. A vector system for delivering GON4L, OTX2 and NEUROD1 to early stage cells; and b. Reagents for detecting induced photoreceptor cells generated from the initial cells, selected from (i) a photoreceptor-specific reporter system, (ii) antibodies for detecting photoreceptor marker proteins, and / or (iii) primers for detecting OTX, CRX, RCVRN, RHO, OPN1 SW, OPN1 MW, and / or OPN1 LW mRNA by PCR; Includes a kit.

15. 13. An expression vector system for a method of producing photoreceptor cells derived from initial cells by the method of any one of claims 1 to 12, wherein the expression vector system comprises one or more nucleic acid sequences operably linked to one or more promoters, the sequences encoding transcription factors (TFs) including at least GON4L, OTX2, and NEUROD1.

16. A combination of transcription factors suitable for producing photoreceptor cells derived from initial cells by the method of any one of claims 1 to 12, wherein the combination of transcription factors comprises at least GON4L, OTX2 and NEUROD1.

Images