The process for producing cone photoreceptor cells

Abstract

The present invention relates to methods and compositions for converting one cell type to another cell type in vitro or in vivo. In particular, the present invention relates to transdifferentiation of a cell to a cone photoreceptor cell. In one aspect, the present invention provides a method for reprogramming a source cell, the method comprising increasing the protein expression of one or more transcription factors, or biologically active fragments or variants thereof, in the source cell, the source cell being reprogrammed to exhibit at least one characteristic of a target cell, the source cell being a glial cell, the target cell being a cone photoreceptor cell or a cone-like photoreceptor cell, and the transcription factor being one or more selected from NEUROG2, CRX, RAX, RORA, NEUROD1, OTX2, ASCL1, PAX6, THRB, MEF2C, FOXP1, and ONECUT1.

Description

[Technical field]

[0001] The present invention relates to methods and compositions for converting one cell type into another cell type in vitro or in vivo. In particular, the present invention relates to the transdifferentiation of certain cells into cone photoreceptor cells.

[0002] Related Applications This application claims priority from Australian Provisional Application No. 2021 / 904199, the entire contents of which are incorporated herein by reference in their entirety. [Background technology]

[0003] Cell-based regenerative therapies require the generation of specific cell types to replace tissues damaged by injury, disease, or aging. Embryonic stem cells (ESCs) have the potential to differentiate in all cell types from the (human) body and therefore have been widely investigated as a source of replacement therapy. However, ESCs cannot be derived in a patient-specific manner because they are established from cultured blastocysts. Thus, immune rejection and ethical concerns are the main barriers preventing the translation of ESC technology, especially human ESC technology, into clinical applications.

[0004] Cell replacement therapy has the potential to rapidly generate a variety of therapeutically important cell types directly from one's own easily accessible tissues, such as skin or blood. Such immunologically compatible cells would also have a low risk of rejection after transplantation. Furthermore, these cells would be terminally differentiated and therefore would exhibit less tumorigenicity.

[0005] Transdifferentiation, the process of converting one cell type to another without going through a pluripotent state, may hold great promise for regenerative medicine but has not yet been reliably applied. Although it may be possible to switch the phenotype of one somatic cell type to another, identifying the factors required for conversion is difficult and in most cases unknown. Identifying factors that directly reprogram cell type identity is currently limited by, among other things, the cost of exhaustive experimental testing of a set of potential factors, an approach that is inefficient and non-scalable.

[0006] Photoreceptor cells, also known simply as photoreceptors, are the light-sensing cells in the retina that form the basis of human vision. Cone cells, or cones, are the photoreceptor cells in the retina of vertebrate eyes, including the human eye. They respond differently to different wavelengths of light and therefore function best in relatively bright light, in contrast to rod cells, which are responsible for color vision and function better in dim light.

[0007] Photoreceptor degeneration is a central feature of many blinding diseases, including retinitis pigmentosa, age-related macular degeneration, choroideremia, and diabetic retinopathy. These diseases affect millions of people worldwide and create a significant socio-economic burden on our healthcare system. Importantly, once the photoreceptors in the eye are lost, there is no cure for blindness. Also, in the later stages of these retinal degenerative diseases, there are often insufficient remaining photoreceptors that can be targeted for pharmacological treatment. In this light, regenerative medicine represents a very attractive approach to address this problem.

[0008] There is a need for new and / or improved methods for generating cells and cell populations, particularly cone photoreceptor cells, for use in research and therapeutic applications.

[0009] The reference to any prior art in this specification is not an admission or suggestion that this prior art forms part of the common knowledge in any jurisdiction, or that this prior art would be reasonably expected to be understood, considered relevant, and / or combined with other prior art by a person skilled in the art. Summary of the Invention

[0010] The present invention relates to in vitro, ex vivo or in vivo methods and compositions for the direct reprogramming of source cells (ie, transdifferentiation or cellular reprogramming) into cells having characteristics of cone photoreceptor cells.

[0011] In one aspect, the present invention provides a method for reprogramming a source cell, the method comprising increasing protein expression of one or more transcription factors, or biologically active fragments or variants thereof, in a source cell, wherein the source cell is reprogrammed to exhibit at least one characteristic of a target cell; the source cells are glial cells; the target cells are cone or cone-like photoreceptor cells; the transcription factor is one or more selected from NEUROG2, CRX, RAX, RORA, NEUROD1, OTX2, ASCL1, PAX6, THRB, MEF2C, FOXP1 and ONECUT1.

[0012] In one aspect, the present invention provides an in vitro, ex vivo, or in vivo method for reprogramming a source cell, the method comprising increasing protein expression of one or more transcription factors, or biologically active fragments or variants thereof, in a source cell, wherein the source cell is reprogrammed to exhibit at least one characteristic of a target cell; the source cells are glial cells; the target cells are cone or cone-like photoreceptor cells; the transcription factor is one or more selected from NEUROG2, CRX, RAX, RORA, NEUROD1, OTX2, ASCL1, PAX6, THRB, MEF2C, FOXP1 and ONECUT1.

[0013] Preferably, the glial cells are selected from the group consisting of Müller glial (MG) cells, astrocytes and microglia. The glial cells may be retinal glial cells.

[0014] Cone photoreceptor cells can be L (or long), M (or medium), or S (or short) type. The methods described herein can generate one or more of these types of cone photoreceptor cells.

[0015] In another aspect, the invention provides a method for generating a cell exhibiting at least one characteristic of a cone photoreceptor cell from a source cell, the method comprising: - increasing the amount of one or more transcription factors, or biologically active fragments or variants thereof, in the source cell; - culturing the source cells for a time and under conditions sufficient to permit differentiation into cone photoreceptor cells, thereby generating from the source cells cells that exhibit at least one characteristic of cone photoreceptor cells; the source cells are glial cells; the transcription factor is one or more selected from NEUROG2, CRX, RAX, RORA, NEUROD1, OTX2, ASCL1, PAX6, THRB, MEF2C, FOXP1 and ONECUT1.

[0016] In any aspect or embodiment, the glial cells are selected from the group consisting of Muller glial (MG) cells, astrocytes, and microglia. The glial cells may be retinal glial cells.

[0017] In another aspect, the invention provides a method for reprogramming a source cell into a cell exhibiting at least one characteristic of a cone photoreceptor cell, the method comprising: - providing a source cell or a cell population comprising source cells; - transfecting said source cells with one or more nucleic acids comprising a nucleotide sequence encoding one or more transcription factors; - culturing said cell or cell population and, optionally, monitoring said cell or cell population for at least one characteristic of a cone photoreceptor cell, the source cells are glial cells; the transcription factor is one or more selected from NEUROG2, CRX, RAX, RORA, NEUROD1, OTX2, ASCL1, PAX6, THRB, MEF2C, FOXP1 and ONECUT1.

[0018] Still further, the present invention provides an in vitro, ex vivo, or in vivo method for reprogramming a source cell into a cell exhibiting at least one characteristic of a cone photoreceptor cell, the method comprising: - providing a source cell or a cell population comprising source cells; - transfecting said source cells with one or more nucleic acids to increase the expression of one or more genes encoding one or more transcription factors; - culturing said cell or cell population and, optionally, monitoring said cell or cell population for at least one characteristic of a cone photoreceptor cell, the source cells are glial cells; the transcription factor is one or more selected from NEUROG2, CRX, RAX, RORA, NEUROD1, OTX2, ASCL1, PAX6, THRB, MEF2C, FOXP1 and ONECUT1.

[0019] Preferably, one or more nucleic acids comprising sgRNA for use in a CRISPR activation system to increase expression of a gene encoding a transcription factor. The sgRNA can be any sgRNA for increasing expression of one or more of NEUROG2, CRX, RAX, RORA, NEUROD1, OTX2, ASCL1, PAX6, THRB, MEF2C, FOXP1 and ONECUT1. The sgRNA can be one or more of those described herein.

[0020] In any aspect of the invention, the glial cells are selected from the group consisting of Muller glial (MG) cells, astrocytes and microglia.

[0021] In any aspect, the method further comprises: determining whether the source cells are expressing at least two of NEUROG2, CRX, RAX, RORA, NEUROD1, OTX2, ASCL1, PAX6, THRB, MEF2C, FOXP1, and ONECUT1; at least three of NEUROG2, CRX, RAX, RORA, NEUROD1, OTX2, ASCL1, PAX6, THRB, MEF2C, FOXP1, and ONECUT1; The method includes transfecting with or to increase expression of nucleic acids encoding at least four of NEUROG2, CRX, RAX, RORA, NEUROD1, OTX2, ASCL1, PAX6, THRB, MEF2C, FOXP1 and ONECUT1, at least five of NEUROG2, CRX, RAX, RORA, NEUROD1, OTX2, ASCL1, PAX6, THRB, MEF2C, FOXP1 and ONECUT1, and at least six of NEUROG2, CRX, RAX, RORA, NEUROD1, OTX2, ASCL1, PAX6, THRB, MEF2C, FOXP1 and ONECUT1.

[0022] In any of the embodiments of the methods of the invention described herein, the source cell is a glial cell (e.g., a Müller glial cell) and the transcription factor, biologically active fragment or variant thereof is (a) ASCL1, CRX and ONECUT1, (b) ASCL1, CRX and THRB, (c) ASCL1, CRX and RORA, (d) ASCL1, CRX and NEUROD1, (e) ASCL1, CRX, and MEF2C; (f) ASCL1, OTX2 and PAX6, (g) CRX, OTX2 and RAX, (h) NEUROD1, NEUROG2 and PAX6, (i) ASCL1, CRX and OTX2, (j) ASCL1, NEUROG2, and OTX2; (k) CRX, NEUROD1 and THRB, (l) OTX2, RAX, and PAX6; (m) ASCL1, NEUROD1, and OTX2; (n) MEF2C, RAX and THRB, (o) MEF2C, PAX6 and OTX2, (p)MEF2C, OTX2 and THRB, (q) MEF2C, OTX2 and RAX, (r) ASCL1, CRX and FOXP1, (s) CRX, NEUROG2, THRB and RAX, (t) ASCL1, CRX, MEF2C, NEUROD1, OTX2 and THRB, (u) MEF2C, PAX6 and THRB, (v) MEF2C, NEUROD1 and PAX6, (w) ASCL1, OTX2 and RAX, (x) ASCL1, NEUROG2 and PAX6, (y) ASCL1, CRX and RAX, (z) CRX, NEUROD1 and OTX2, (aa) CRX, NEUROG2 and OTX2, (bb) ASCL1, CRX and NEUROG2, (cc) CRX, RORA and THRB, (dd) NEUROD1, OTX2 and RAX, (ee) CRX, RAX and THRB, (ff) MEF2C, OTX2 and RORA, (gg) NEUROG2, PAX6 and RAX, (hh) ASCL1, CRX and PAX6, (ii) FOXP1, NEUROG2, PAX6 and THRB, (jj) CRX, NEUROD1 and RAX, (kk)CRX, NEUROG2 and PAX6, (ll) CRX, NEUROD1, OTX2 and RAX, (mm) NEUROG2, OTX2 and PAX6, (nn) CRX and RAX, (oo) PAX6 and RAX, (pp)CRX, NEUROG2, OTX2 and RAX, or (qq)NEUROG2 and PAX6.

[0023] In any of the embodiments of the methods of the invention described herein, the source cell is a glial cell (e.g., a Müller glial cell) and the transcription factor, biologically active fragment or variant thereof is (a) ASCL1, CRX and ONECUT1, (b) ASCL1, CRX and THRB, (c) ASCL1, CRX and RORA, (d) ASCL1, CRX and NEUROD1, (e) ASCL1, CRX, and MEF2C; (f) ASCL1, OTX2 and PAX6, (g) CRX, OTX2 and RAX, (h) NEUROD1, NEUROG2 and PAX6, (i) ASCL1, CRX and OTX2, (j) ASCL1, NEUROG2, and OTX2; (k) OTX2, RAX, and PAX6; (l) ASCL1, NEUROD1, and OTX2, (m) MEF2C, RAX and THRB, (n) MEF2C, PAX6 and OTX2, (o) MEF2C, OTX2 and THRB, (p)MEF2C, OTX2 and RAX, (q) ASCL1, CRX and FOXP1, (r) CRX, NEUROG2, THRB and RAX, (s) ASCL1, CRX, MEF2C, NEUROD1, OTX2 and THRB, (t) MEF2C, PAX6 and THRB, (u) MEF2C, NEUROD1 and PAX6, (v) ASCL1, OTX2 and RAX, (w) ASCL1, NEUROG2 and PAX6, (x) ASCL1, CRX and RAX, (y) CRX, NEUROD1 and OTX2, (z) CRX, NEUROG2 and OTX2, (aa) ASCL1, CRX and NEUROG2, (bb) CRX, RORA and THRB, (cc) NEUROD1, OTX2 and RAX, (dd) CRX, RAX and THRB, (ee) MEF2C, OTX2 and RORA, (ff) NEUROG2, PAX6 and RAX, (gg) ASCL1, CRX and PAX6, (hh) FOXP1, NEUROG2, PAX6 and THRB, (ii) CRX, NEUROD1 and RAX, (jj) CRX, NEUROG2 and PAX6, (kk) CRX, NEUROD1, OTX2 and RAX, (ll) NEUROG2, OTX2 and PAX6, (mm) CRX and RAX, (nn)PAX6 and RAX, (oo) CRX, NEUROG2, OTX2 and RAX, or (pp)NEUROG2 and PAX6.

[0024] In any of the embodiments of the methods of the invention described herein, the source cell is a glial cell (e.g., a Müller glial cell) and the transcription factor, biologically active fragment or variant thereof is (a) ASCL1, CRX and ONECUT1, (b) ASCL1, CRX and THRB, (c) ASCL1, CRX and RORA, (d) ASCL1, CRX and NEUROD1, (e) ASCL1, CRX, and MEF2C; (f) ASCL1, OTX2 and PAX6, (g) CRX, OTX2 and RAX, (h) NEUROD1, NEUROG2 and PAX6, (i) ASCL1, CRX and OTX2, (j) ASCL1, NEUROG2, and OTX2; (k) CRX, NEUROD1 and THRB, (l) OTX2, RAX, and PAX6; (m) ASCL1, NEUROD1, and OTX2; (n) MEF2C, RAX and THRB, (o) MEF2C, PAX6 and OTX2, (p)MEF2C, OTX2 and THRB, (q) MEF2C, OTX2 and RAX, (r) ASCL1, CRX and FOXP1, (s) CRX, NEUROG2, THRB and RAX, (t) ASCL1, CRX, MEF2C, NEUROD1, OTX2 and THRB, (u) MEF2C, PAX6 and THRB, (v) MEF2C, NEUROD1 and PAX6, (w) ASCL1, OTX2 and RAX, (x) ASCL1, NEUROG2 and PAX6, (y) ASCL1, CRX and RAX, (z) CRX, NEUROD1 and OTX2, (aa) CRX, NEUROG2 and OTX2, (bb) ASCL1, CRX and NEUROG2, (cc) CRX, RORA and THRB, (dd) NEUROD1, OTX2 and RAX, or (ee) CRX, RAX and THRB.

[0025] In any of the embodiments of the methods of the invention described herein, the source cell is a glial cell (e.g., a Müller glial cell) and the transcription factor, biologically active fragment or variant thereof is (a) ASCL1, NEUROG2 and OTX2, (b) CRX, NEUROG2, THRB and RAX, (c) NEUROD1, NEUROG2 and PAX6, (d) NEUROG2 and PAX6, (e) ASCL1, OTX2 and PAX6, (f) CRX, NEUROD1 and THRB, (g) ASCL1, CRX and RORA, (h) ASCL1, CRX and ONECUT1, (i) CRX, OTX2 and RAX, (j) ASCL1, CRX and NEUROD1, (k) ASCL1, NEURGOG2 and PAX6, (l) ASCL1, CRX and NEUROG2, (m) ASCL1, CRX and THRB, (n) ASCL1, CRX and MEF2C, (o) CRX, NEUROG2, OTX2 and RAX, (p) ASCL1, CRX, MEF2C, OTX2, and THRB, (q) CRX, NEURGOD1 and OTX2, (r) OTX2; RAX and PAX6, (s) ASCL1; NEUROD1 and OTX2, (t)CRX;NEUROG2 and OTX2, (u) ASCL1; CRX and OTX2, (v) ASCL1, CRX and RAX, (w) CRX, RORA and THRB, (x) CRX, RAX and THRB, or (y) NEUROD1; OTX2 and RAX.

[0026] In any of the embodiments of the methods of the invention described herein, the source cell is a glial cell (e.g., a Müller glial cell) and the transcription factor, biologically active fragment or variant thereof is (a) ASCL1, CRX and ONECUT1, (b) ASCL1, CRX and THRB, (c) ASCL1, CRX and RORA, (d) ASCL1, CRX and NEUROD1, (e) ASCL1, CRX, and MEF2C; (f) ASCL1, OTX2 and PAX6, (g) CRX, OTX2 and RAX, (h) NEUROD1, NEUROG2 and PAX6, (i) ASCL1, CRX and OTX2, (j) ASCL1, NEUROG2, and OTX2; (k) CRX, NEUROD1 and THRB, (l) OTX2, RAX, and PAX6; (m) ASCL1, NEUROD1, and OTX2; (n) MEF2C, RAX and THRB, (o) MEF2C, PAX6 and OTX2, (p)MEF2C, OTX2 and THRB, (q) MEF2C, OTX2 and RAX, (r) ASCL1, CRX and FOXP1, (s) CRX, NEUROG2, THRB and RAX, (t) ASCL1, CRX, MEF2C, NEUROD1, OTX2 and THRB, (u) MEF2C, PAX6 and THRB, (v) MEF2C, NEUROD1 and PAX6, (w) ASCL1, OTX2 and RAX, or (x) ASCL1, NEUROG2 and PAX6.

[0027] In any of the embodiments of the methods of the invention described herein, the source cell is a glial cell (e.g., a Müller glial cell) and the transcription factor, biologically active fragment or variant thereof is (a) ASCL1, NEUROG2 and OTX2, (b) CRX, NEUROG2, THRB and RAX, (c) NEUROD1, NEUROG2 and PAX6, (d) NEUROG2 and PAX6, (e) ASCL1, OTX2 and PAX6, (f) CRX, NEUROD1 and THRB, (g) ASCL1, CRX and RORA, (h) ASCL1, CRX and ONECUT1, (i) CRX, OTX2 and RAX, (j) ASCL1, CRX and NEUROD1, (k) ASCL1, NEURGOG2 and PAX6, (l) ASCL1, CRX and NEUROG2, or (m) ASCL1, CRX and THRB.

[0028] In any of the embodiments of the methods of the invention described herein, the source cell is a glial cell (e.g., a Müller glial cell) and the transcription factor, biologically active fragment or variant thereof is (a) ASCL1, CRX and ONECUT1, (b) ASCL1, CRX and THRB, (c) ASCL1, CRX and RORA, (d) ASCL1, CRX and NEUROD1, (e) ASCL1, CRX, and MEF2C; (f) ASCL1, OTX2 and PAX6, (g) CRX, OTX2 and RAX, (h) NEUROD1, NEUROG2 and PAX6, (i) ASCL1, CRX and OTX2, (j) ASCL1, NEUROG2, and OTX2; (k) CRX, NEUROD1 and THRB, (l) OTX2, RAX, and PAX6; (m) ASCL1, NEUROD1, and OTX2; (n) MEF2C, RAX and THRB, (o) MEF2C, PAX6 and OTX2, (p)MEF2C, OTX2 and THRB, (q) MEF2C, OTX2 and RAX, or (r) ASCL1, CRX and FOXP1.

[0029] In any of the embodiments of the methods of the invention described herein, the source cell is a glial cell (e.g., a Müller glial cell) and the transcription factor, biologically active fragment or variant thereof is (a) ASCL1, NEUROG2 and OTX2, (b) CRX, NEUROG2, THRB and RAX, (c) NEUROD1, NEUROG2 and PAX6, (d) NEUROG2 and PAX6, (e) ASCL1, OTX2, and PAX6, or (f) CRX, NEUROD1 and THRB.

[0030] In any of the aspects of the methods of the invention described herein, the source cell is a Müller glial cell and the transcription factor, biologically active fragment or variant thereof is (a) ASCL1, CRX and ONECUT1, (b) ASCL1, CRX and THRB, (c) ASCL1, CRX and RORA, (d) ASCL1, CRX and NEUROD1, (e) ASCL1, CRX, and MEF2C; (f) ASCL1, OTX2 and PAX6, (g) CRX, OTX2 and RAX, (h) NEUROD1, NEUROG2 and PAX6, (i) ASCL1, CRX and OTX2, (j) ASCL1, NEUROG2, and OTX2, or (k) CRX, NEUROD1 and THRB.

[0031] In any of the embodiments of the methods of the invention described herein, the source cell is a glial cell (e.g., a Müller glial cell) and the transcription factor, biologically active fragment or variant thereof is (a) ASCL1, CRX and ONECUT1, (b) ASCL1, CRX and THRB, (c) ASCL1, CRX and RORA, (d) ASCL1, CRX and NEUROD1, (e) ASCL1, CRX, and MEF2C; (f) ASCL1, OTX2 and PAX6, (g) CRX, OTX2 and RAX, (h) NEUROD1, NEUROG2 and PAX6, or (i) ASCL1, CRX and OTX2.

[0032] In any of the embodiments of the methods of the invention described herein, the source cell is a glial cell (e.g., a Müller glial cell) and the transcription factor, biologically active fragment or variant thereof is (a) ASCL1, CRX and ONECUT1, (b) ASCL1, CRX and THRB, (c) ASCL1, CRX and RORA, (d) ASCL1, CRX and NEUROD1, (e) ASCL1, CRX, and MEF2C, or (f) ASCL1, OTX2 and PAX6.

[0033] In any of the embodiments of the methods of the invention described herein, the source cell is a glial cell (e.g., a Müller glial cell) and the transcription factor, biologically active fragment or variant thereof is (a) ASCL1, CRX and ONECUT1, (b) ASCL1, CRX and THRB, (c) ASCL1, CRX and RORA, (d) ASCL1, CRX and NEUROD1, or (e) ASCL1, CRX, and MEF2C.

[0034] In any of the embodiments of the methods of the invention described herein, the source cell is a glial cell (e.g., a Müller glial cell) and the transcription factor, biologically active fragment or variant thereof is (a) ASCL1, CRX and ONECUT1, (b) ASCL1, OTX2 and PAX6, or (c) ASCL1, CRX and RORA.

[0035] In any of the embodiments of the methods of the invention described herein, the source cell is a glial cell (e.g., a Müller glial cell) and the transcription factor, biologically active fragment or variant thereof is (a) ASCL1, CRX and ONECUT1, (b) ASCL1, CRX and THRB, or (c) ASCL1, CRX and RORA.

[0036] In any of the embodiments of the methods of the invention described herein, the source cell is a glial cell (e.g., a Müller glial cell) and the transcription factor, biologically active fragment or variant thereof is (a) ASCL1, NEUROG2 and OTX2, or (b) CRX, NEUROG2, THRB and RAX.

[0037] In any of the embodiments of the methods of the invention described herein, the source cell is a glial cell (e.g., a Müller glial cell) and the transcription factor, biologically active fragment or variant thereof is (a) ASCL1, CRX and ONECUT1, or (b) ASCL1, CRX and THRB.

[0038] Preferably, the at least one characteristic of a cone photoreceptor cell is upregulation of any one or more target cell markers and / or a change in cell morphology. Relevant markers are described herein and known in the art. Exemplary markers for cone photoreceptor cells include: -ARR3, CNGB3, GNAT2, GNGT2, GRK7, GUCA1C, PDE6C, PDE6H, RXRG, THRB, OPN1LW, OPN1MW and OPN1SW, For example, electrophysiological responses under photopic conditions, as described in the Examples.

[0039] In any embodiment, the cone photoreceptor cells, or cone photoreceptor-like cells produced or generated from the methods or uses described herein exhibit detectable levels of any one, two, three, four, five, six, seven, eight, nine, ten, eleven or twelve of the following markers: ARR3, CNGB3, GNAT2, GNGT2, GRK7, GUCAlC, PDE6C, PDE6H, RXRG, THRB, OPN1LW, OPN1MW and OPN1SW. Cone photoreceptor cells, or cone photoreceptor-like cells may have detectable levels of OPN1LW, OPN1MW or OPN1SW.

[0040] Additional examples of photoreceptor markers include opsins, which are light-detecting molecules, such as rhodopsin (rod photoreceptor cells), red / green opsin (cone receptor cells), blue opsin (cone receptor cells), and recoverin (rod photoreceptor cells, cone receptor cells).

[0041] In any embodiment, a combination of transcription factors selected from one or more of those selected from NEUROG2, CRX, RAX, RORA, NEUROD1, OTX2, ASCL1, PAX6, THRB, MEF2C, FOXP1, and ONECUT1 results in cone photoreceptor cells, or cone photoreceptor-like cells, with a fold change in opsin mRNA expression that is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 fold or greater compared to opsin expression in the source cell type. Preferably, the fold increase is 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 fold or greater compared to opsin expression in the source cell type. Preferably, the fold increase is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 fold or more compared to opsin expression in the source cell type. Preferably, the fold increase is 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 fold or more compared to opsin expression in the source cell type. Preferably, the fold increase is 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 fold or more compared to opsin expression in the source cell type. Preferably, the fold increase is 4, 5, 6, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 fold or more compared to opsin expression in the source cell type. Preferably, the fold increase is 10, 11, 12, 13, 14, 15, 16, 17, or 18 fold or more compared to opsin expression in the source cell type. Most preferably, the fold increase is greater than or equal to 10, 11, 12, 13, 14, 15, 16, 17, or 18 fold or more compared to opsin expression in the source cell type, preferably, the fold increase is greater than or equal to 13, 14, 15, 16, 17, or 18 fold or more compared to opsin expression in the source cell type. The opsins can be OPN1LW / MW and / or OPN1SW.

[0042] As used herein, an opsin can be encoded by the gene OPN1LW / MW or the gene OPN1SW.

[0043] In any aspect of the invention, the source cell is a human cell. When the source cell is a Muller glial cell, it can be a human Muller glial cell.

[0044] Typically, suitable conditions for photoreceptor cell differentiation include culturing the cells in a suitable medium for a sufficient period of time, which may be at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 days. A suitable medium may be one as shown in Table 2.

[0045] In any aspect of the invention, the cells may be contacted with trichostatin A during the transfection step or the culture step.

[0046] In another aspect, the invention also provides a cell exhibiting at least one characteristic of a cone photoreceptor cell produced by the methods described herein.

[0047] In any of the methods described herein, the method may further include expanding the cells exhibiting at least one characteristic of cone photoreceptor cells to increase the proportion of cells in the population exhibiting at least one characteristic of cone photoreceptor cells. Expanding the cells may be performed in culture for a time and under conditions sufficient to generate a population of cells as described below.

[0048] In any of the methods described herein, the method may further comprise administering to the individual a cell, or a cell population comprising a cell, that exhibits at least one characteristic of a cone photoreceptor cell.

[0049] The invention also provides a population of cells, wherein at least 0.01%, at least 0.02%, at least 0.03%, at least 0.04%, at least 0.05, at least 0.06%, at least 0.07%, at least 0.08%, at least 0.09%, at least 0.1%, at least 0.15%, at least 0.2%, at least 0.25%, at least 0.3%, at least 0.35%, at least 0.4%, at least 0.45%, at least 0.5, at least 0.6%, at least 0.7%, at least 0.8%, at least 0.9%, at least 1%, at least 2%, at least 3%, at least 4%, or at least 5% of the cells exhibit at least one characteristic of cone photoreceptor cells, and wherein the cells are produced by the methods described herein. Preferably, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or at least 100% of the cells in the population exhibit at least one characteristic of cone photoreceptor cells. The invention also provides a population of cells, wherein 0.01%, 0.02%, 0.03%, 0.04%, 0.05, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.15%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4% or 5% of the cells in the population exhibit at least one characteristic of a cone photoreceptor cell, and wherein the cells are produced by the methods described herein.Preferably, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% of the cells in the population exhibit at least one characteristic of cone photoreceptor cells.

[0050] The present invention also relates to a kit for producing a cell exhibiting at least one characteristic of a cone photoreceptor cell disclosed herein. In some embodiments, the kit comprises one or more nucleic acids having one or more nucleic acid sequences encoding a transcription factor or biologically active fragment variants thereof as described herein, including the specific combinations referred to in (a)-(ee) herein. Preferably, the kit can be used with a source cell as referred to herein. In some embodiments, the kit further comprises instructions for reprogramming the source cell into a cell exhibiting at least one characteristic of a cone photoreceptor cell according to the methods disclosed herein. Preferably, the present invention provides a kit when used in the methods of the present invention described herein.

[0051] In another aspect, the present invention relates to a composition comprising at least one source cell as described herein and at least one agent that increases expression of genes encoding one or more transcription factors in the source cell. Further, the transcription factor can be any one or more of those described herein, including combinations of those referenced in (a)-(ee) herein.

[0052] Typically, gene expression or amount of the transcription factors described herein is increased by contacting the cell with an agent that increases the expression of the transcription factor. Preferably, the agent is selected from the group consisting of nucleotide sequences, proteins, aptamers and small molecules, ribosomes, RNAi agents and peptide nucleic acids (PNAs), and analogs or biologically active fragments or variants thereof. Preferably, the agent is exogenous. In a preferred embodiment, one or more agents are transgenes or CRISPR components such as those described herein that induce endogenous gene activation. For example, CRISPR activation systems and their components, including sgRNAs such as those described herein, are contemplated as agents that increase the expression of one or more transcription factors.

[0053] Typically, gene expression or amount of a transcription factor described herein is increased by introducing into a cell at least one nucleic acid comprising a nucleotide sequence encoding the transcription factor or encoding a functional fragment thereof. Preferably, the nucleotide sequence encoding the transcription factor is at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a sequence having an accession number listed in Table 1.

[0054] Gene expression, or the amount, of a transcription factor described herein can also be increased by introducing at least one nucleic acid (such as an sgRNA) for use in a CRISPR activation system to increase expression of the gene encoding the transcription factor.

[0055] Preferably, the nucleic acid further comprises a heterologous promoter. Preferably, the nucleic acid is in a vector, such as a viral vector or a non-viral vector. In one embodiment, the nucleic acid can be RNA, preferably mRNA, most preferably synthetic mRNA. Preferably, the vector is a viral vector that comprises a genome that is not integrated into the host cell genome. The viral vector can be a retroviral vector, an AAV vector, a baculoviral vector, or a lentiviral vector.

[0056] In another aspect, the invention relates to a nucleic acid as described herein, or a vector comprising a nucleic acid, which may comprise one or more nucleotide sequences encoding one or more of the transcription factors described herein. Preferably, the nucleic acid or vector encodes one or more of the set of transcription factors described herein, including (a)-(ee) above, and in Table 3 below. In one embodiment, the nucleic acid or vector comprises one or more of the sequences set forth above in Table 1, or a sequence encoding any one or more of the amino acid sequences listed in Table 1. In another embodiment, the nucleic acid or vector is any one described herein.

[0057] In another aspect, the present invention relates to a CRISPR activation system for increasing expression of a gene encoding one or more of the transcription factors described herein. Preferably, the CRISPR activation system results in increased expression of one or more of the set of transcription factors described herein, including (a)-(qq) above and Table 3 below. In one embodiment, the CRISPR activation system comprises an sgRNA as described herein, including an sgRNA targeting a gene selected from the group consisting of CRX, MEF2C, THRB, RAX, NEUROD1, RORA, OTX2, NEUROG2 / NGN2, PAX6, FOXP1, ASCL1, and ONECUT1 (On).

[0058] In another aspect, the invention relates to an in vitro or ex vivo cell comprising a nucleic acid or vector of the invention as described herein.

[0059] In any aspect of the invention, the methods described herein may have one or more, or all, steps carried out in vitro, ex vivo or in vivo.

[0060] In another aspect, the invention provides a method of treating a condition associated with or caused by degeneration or loss of cone photoreceptor cells in an individual in need thereof, the method comprising administering to the individual a cell or population of cells generated in vitro or ex vivo by any of the methods described herein.

[0061] In another aspect, the invention provides the use of a cell or population of cells generated in vitro or ex vivo by any of the methods described herein in the manufacture of a medicament for treating a condition associated with or caused by degeneration or loss of cone photoreceptor cells in an individual in need thereof.

[0062] In another aspect, the invention provides a cell or cell population produced in vitro or ex vivo by any of the methods described herein for use in an individual in need of treatment for a condition associated with or caused by degeneration or loss of cone photoreceptor cells.

[0063] In any embodiment, the nucleic acid or vector comprises or consists of an expression construct. In some embodiments, the expression construct comprises one of more features of AAV, rAAV, lentivirus or baculovirus vectors or synthetic mRNA. Preferably, the expression construct comprises one or more features of the AAV vector or rAAV vector of the present invention described herein.

[0064] In another aspect, the present invention also provides a recombinant vector comprising an expression construct described herein. The recombinant vector may be a recombinant AAV (rAAV) vector.

[0065] A promoter in the expression constructs described herein or any other embodiment of the invention can be any nucleotide sequence that can induce RNA polymerase to bind to and transcribe a coding sequence. The promoter can be a ubiquitous promoter or a glial cell-specific promoter.

[0066] In a preferred embodiment, the promoter is a CAG promoter. The CAG promoter preferably comprises a cytomegalovirus (CMV) early enhancer element, a promoter, the first exon and the first intron of the chicken beta-actin (CBA) gene, and a splice acceptor of the rabbit beta-globin gene.

[0067] In one embodiment, the nucleotide sequence encoding the CMV early enhancer element is 245 bp in length and is referred to herein as SEQ ID NO: 1. Preferably, the CMV early enhancer element comprises a nucleotide sequence substantially as set forth in SEQ ID NO: 1, or a fragment or variant thereof.

[0068] In one embodiment, the nucleotide sequence encoding the GFAP promoter is 681 bp in length and is referred to herein as SEQ ID NO: 2. Preferably, the promoter comprises a nucleotide sequence substantially as set out in SEQ ID NO: 2, or a fragment or variant thereof.

[0069] In one embodiment, the nucleotide sequence encoding the first intron of the chicken beta-actin gene (CBA) is 408 bp in length and is referred to herein as SEQ ID NO: 3. Preferably, the first intron of CBA comprises a nucleotide sequence substantially as set forth in SEQ ID NO: 3, or a fragment or variant thereof.

[0070] Preferably, an expression construct of the invention described herein, or any other embodiment, comprises a nucleotide sequence encoding a Kozak sequence that enhances transcription factor expression, or a biologically active fragment or variant thereof. Preferably, the Kozak coding sequence is located 5' of a nucleotide sequence encoding one or more of the set of transcription factors described herein, including (a)-(qq) above, and Table 3 below, or a biologically active fragment or variant thereof.

[0071] In one embodiment, the nucleotide sequence encoding the Kozak sequence is 10 bp in length and is referred to herein as SEQ ID NO: 4. Preferably, the Kozak sequence comprises a nucleotide sequence substantially as set forth in SEQ ID NO: 4, or a fragment or variant thereof.

[0072] Preferably, an expression construct of the invention described herein, or any other embodiment, comprises a nucleotide sequence encoding a Woodchuck Hepatitis Virus Post-Transcriptional Regulatory Element (WPRE) that enhances expression of one or more of the set of transcription factors described herein, including (a)-(qq) above, and Table 3 below, or biologically active fragments or variants thereof. Preferably, the WPRE coding sequence is located 3' to the nucleotide sequence encoding one or more of the set of transcription factors described herein, including (a)-(qq) above, and Table 3 below, or biologically active fragments or variants thereof.

[0073] In one embodiment, the nucleotide sequence encoding the WPRE is 593 bp in length and is referred to herein as SEQ ID NO: 5. Preferably, the WPRE comprises a nucleotide sequence substantially as set forth in SEQ ID NO: 5, or a fragment or variant thereof.

[0074] Preferably, an expression construct of the invention described herein, or any other embodiment, comprises a nucleotide sequence encoding a bovine growth hormone (bGH) polyA tail. Preferably, the bGH polyA tail coding sequence is located 3' to a nucleotide sequence encoding one or more of the set of transcription factors described herein, including (a)-(qq) above, and Table 3 below, or biologically active fragments or variants thereof, preferably 3' to a WPRE coding sequence.

[0075] In one embodiment, the nucleotide sequence encoding the bovine growth hormone (bGH) polyA tail is 269 bp in length and is referred to herein as SEQ ID NO: 6. Preferably, the bovine growth hormone polyA tail comprises a nucleotide sequence substantially as set forth in SEQ ID NO: 6, or a fragment or variant thereof.

[0076] In any embodiment, the expression construct comprises AAV inverted terminal repeats (ITRs), e.g., AAV ITRs flanking a nucleotide sequence encoding one or more of the set of transcription factors described herein, including (a)-(qq) above, and Table 3 below, or biologically active fragments or variants thereof.

[0077] Preferably, an expression construct of the invention described herein, or any other embodiment, comprises a left and / or a right ITR, preferably each ITR being located at the 5' and / or 3' end of the construct.

[0078] In one embodiment, the nucleotide sequence of the left ITR is represented herein as SEQ ID NO:7.

[0079] In one embodiment, the nucleotide sequence of the right ITR is represented herein as SEQ ID NO:8.

[0080] In another aspect, the present invention also provides an adeno-associated virus (AAV) vector, lentivirus vector, baculovirus vector, or mRNA (e.g., synthetic mRNA) comprising a nucleotide sequence encoding one or more of the set of transcription factors described herein, including (a)-(qq) above, and in Table 3 below, or a biologically active fragment or variant thereof. Typically, the nucleotide sequence encoding one or more of the set of transcription factors described herein, including (a)-(qq) above, and in Table 3 below, or a biologically active fragment or variant thereof, is flanked by two AAV inverted terminal repeats (ITRs).

[0081] In any embodiment, the AAV vector, lentiviral vector or baculoviral vector is recombinant, synthetic, purified, or substantially purified.

[0082] In some embodiments, the AAV vector is a recombinant AAV (rAAV) vector. The rAAV can be a naturally occurring vector or a vector having a hybrid AAV serotype. The rAAV can be AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, AAV-10, AAV-11, AAV-12, AAV-13, ShH10 and ShH10Y.

[0083] The recombinant AAV vector may be a bioengineered vector. The rAAV may be Anc80, DJ, DJ / 8, KP1, KP2, KP3, LK01, LK02, LK03, LK19, NP6, NP22, NP40, NP59, NP66, NP84, NP94, rh10, 2i8, 7m8, PHP.eB, and AAV2 Retro.

[0084] The recombinant vector may be SYD01, SYD03, SYD09, HRS1, HRS19, HRS5, CD15, T33, CMRI-01, CMRI-02, CMRI-03, CMRI-04, CMRI-05, CMRI-06, CMRI-07 and CMRI-08.

[0085] However, preferably the rAAV is ShH10 or ShH10Y.

[0086] Advantageously, ShH10 and ShH10Y, derived from the AAV6 parental serotype, allow efficient and selective Müller cell infection via intravitreal injection. ShH10 and ShH10Y also show significantly improved transduction compared to AAV2 (>60%) and AAV6.

[0087] As used herein, the term "recombinant (rAAV) vector" means a recombinant AAV-derived nucleic acid that contains at least one long terminal repeat sequence.

[0088] Preferably, an expression construct, recombinant plasmid vector or any other embodiment of the invention described herein comprises at least one stuffer sequence, preferably one or more of the following stuffer sequences described below: Preferably, the recombinant plasmid vector comprises a first, second, third, fourth, fifth, sixth, and / or seventh stuffer sequence, preferably the first, second, third, fourth, fifth, sixth, and seventh stuffer sequences are as described herein (e.g., SEQ ID NOs: 9-15).

[0089] In one embodiment, the first stuffer sequence is represented herein as SEQ ID NO:9.

[0090] In one embodiment, the second stuffer sequence is represented herein as SEQ ID NO:10.

[0091] In one embodiment, the third stuffer sequence is represented herein as SEQ ID NO:11.

[0092] In one embodiment, the fourth stuffer sequence is represented herein as SEQ ID NO:12.

[0093] In one embodiment, the fifth stuffer sequence is represented herein as SEQ ID NO:13.

[0094] In one embodiment, the sixth stuffer sequence is represented herein as SEQ ID NO:14.

[0095] In one embodiment, the sixth stuffer sequence is represented herein as SEQ ID NO:15.

[0096] Preferably, the expression construct, recombinant plasmid vector or any other embodiment of the invention described herein comprises an antibiotic resistance gene. Typically, the antibiotic resistance gene is a nucleotide sequence encoding a kanamycin resistance gene.

[0097] In one embodiment, the nucleotide sequence encoding the kanamycin resistance gene is 816 bp in length and is referred to herein as SEQ ID NO:16.

[0098] Preferably, the expression construct, recombinant plasmid vector or any other embodiment of the invention described herein comprises a pUC origin. Typically, the nucleotide sequence encoding the pUC origin is 668 bp in length and is referred to herein as SEQ ID NO: 17.

[0099] In any embodiment, the AAV vector, lentiviral vector, baculoviral vector, or synthetic mRNA further comprises one or more regulatory sequences (e.g., promoters) that enable or cause expression of one or more of the set of transcription factors described herein, including (a)-(ee) above, and Table 3 below, or biologically active fragments or variants thereof, in glial cells, preferably retinal glial cells.

[0100] Preferably, the promoter is a ubiquitous promoter or a glial cell-specific promoter. In any embodiment, a nucleotide sequence encoding one or more of the set of transcription factors described herein, including (a)-(qq) above and Table 3 below, or biologically active fragments or variants thereof, is operably linked to the promoter.

[0101] An example of a ubiquitous promoter is the CAG promoter. The CAG promoter preferably comprises a cytomegalovirus (CMV) early enhancer element, a promoter, the first exon and the first intron of chicken beta-actin (CBA) gene, and a splice acceptor of rabbit beta-globin gene.

[0102] Examples of glial cell-specific promoters include the promoters of the GFAP, GLAST and RLBP1 genes, and / or combinations of glial cell-specific transcription factor regulatory elements.

[0103] In some embodiments, the AAV vector comprises a CMV promoter, e.g., as described herein. In some embodiments, the AAV vector comprises a Kozak sequence, e.g., as described herein. In some embodiments, the vector comprises one or more ITR sequences flanking the vector protein encoding one or more of the set of transcription factors described herein, e.g., as described herein, including (a)-(qq) above, and Table 3 below, or biologically active fragments or variants thereof. In some embodiments, the vector comprises a polyadenylation sequence. In some embodiments, the vector comprises a selection marker. Preferably, the selection marker is an antibiotic resistance gene, such as an ampicillin resistance gene or a kanamycin resistance gene.

[0104] In any embodiment, the ITR, or each ITR, if there is more than one, is a wild-type AAV ITR sequence, or an ITR, as described herein.

[0105] In one embodiment, the present invention provides a method for the preparation of a nucleic acid sequence comprising, in 5' to 3' order: (a) a 5' AAV ITR, e.g., SEQ ID NO: 7 or 8; (b) a CMV enhancer, e.g., SEQ ID NO:1; (c) glial cell-specific promoter, (d) a transgene encoding one or more of the set of transcription factors described herein, including (a)-(qq) above, and Table 3 below, or biologically active fragments or variants thereof; (e) WPRE, e.g., SEQ ID NO:5, (f) bovine growth hormone polyA signal tail, e.g., SEQ ID NO:6, and (g) Provided is a recombinant adeno-associated virus (AAV) vector comprising a nucleic acid comprising a 3′ AAV ITR, e.g., SEQ ID NO: 7 or 8.

[0106] In another aspect, the present invention provides a recombinant adeno-associated virus (rAAV), comprising: (i) an AAV capsid protein; (ii) a recombinant adeno-associated virus (rAAV) comprising the AAV vector of the present invention described herein.

[0107] In one embodiment, the AAV capsid protein is a ShH10 or ShH10Y capsid protein.

[0108] In any embodiment, the rAAV may be an AAV variant or mutant described herein.

[0109] In another aspect, the present invention provides a pharmaceutical composition comprising an isolated nucleic acid of the invention as described herein, a genetic construct of the invention as described herein, an AAV vector of the invention as described herein, a recombinant AAV of the invention as described herein, and a pharma- ceutically acceptable carrier, diluent or excipient.

[0110] In another aspect, the invention provides a plasmid comprising an isolated nucleic acid comprising an expression construct of the invention as described herein, or an AAV vector of the invention as described herein.

[0111] In another aspect, the present invention provides a baculovirus vector comprising a nucleic acid of the invention as described herein.

[0112] In another aspect, the present invention provides a method for producing a composition comprising: (i) a first vector encoding one of a number of adeno-associated virus rep proteins and / or one or more adeno-associated virus cap proteins; (ii) a second vector comprising a nucleotide sequence encoding one or more of the set of transcription factors described herein, including (a)-(qq) above, and Table 3 below, or a biologically active fragment or variant thereof.

[0113] In one embodiment, the first vector is a plasmid and the second vector is a plasmid, hi another embodiment, the first vector is a baculovirus vector and the second vector is a baculovirus vector.

[0114] Typically, the cell is a mammalian cell, preferably the mammalian cell is a HEK293 cell, or alternatively, the cell is an insect cell, preferably the insect cell is a SF9 cell.

[0115] In another aspect, the present invention provides a method of producing an AAV of the invention described herein, the method comprising: (i) delivering to a cell a recombinant AAV vector comprising a first vector encoding one or more adeno-associated virus rep proteins and / or one or more adeno-associated cap proteins, and an expression cassette comprising a nucleotide sequence encoding one or more of the set of transcription factors described herein, including (a)-(qq) above, and Table 3 below, or biologically active fragments or variants thereof; (ii) culturing the cells under conditions that permit packaging of the AAV; (iii) harvesting the cultured host cells or culture medium for harvesting the AAV.

[0116] In another aspect, the invention provides a method of reducing the progression of or reversing vision loss associated with cone dystrophy in a subject, the method comprising administering to the subject an isolated nucleic acid of the invention as described herein, a genetic construct of the invention as described herein, an AAV vector of the invention as described herein, a recombinant AAV of the invention as described herein, or a pharmaceutical composition of the invention as described herein, thereby reducing the progression of or reversing vision loss associated with or caused by the degeneration or loss of cone photoreceptor cells.

[0117] In another aspect, the invention provides the use of an isolated nucleic acid of the invention as described herein, an AAV vector of the invention as described herein, a recombinant AAV of the invention as described herein, or a pharmaceutical composition of the invention as described herein in the manufacture of a medicament for reducing the progression of or reversing vision loss associated with or caused by degeneration or loss of cone photoreceptor cells in a subject.

[0118] In another aspect, the invention provides an isolated nucleic acid of the invention as described herein, an AAV vector of the invention as described herein, a recombinant AAV of the invention as described herein, or a pharmaceutical composition of the invention as described herein for use in reducing the progression of or restoring vision associated with or caused by degeneration or loss of cone photoreceptor cells in a subject.

[0119] In any embodiment, preferably the subject is a human.

[0120] In any aspect or embodiment, a condition associated with or caused by the degeneration or loss of cone photoreceptor cells may also be referred to as a cone cell disorder, which is associated with or causes a change in vision, typically a decrease in vision.

[0121] In some embodiments, the cone cell disorder is a retinal degenerative disorder. In certain embodiments, the retinal degenerative disorder is selected from the group consisting of achromatopsia, blue-cone monochromacy, protanopia, deutanopia, and tritanopia. In some embodiments, the cone cell disorder is a macular dystrophy or a retinal dystrophy. The macular dystrophy may be selected from the group consisting of Stargardt macular dystrophy, cone dystrophy (including rod-cone dystrophy and cone-rod dystrophy), spinocerebellar ataxia type 7, and Bardet-Biedl syndrome-1. Preferably, the macular dystrophy is Stargardt macular dystrophy or a cone-rod dystrophy. In some embodiments, the cone cell disorder is a central macular vision disorder or a retinal dystrophy. In certain embodiments, the central macular vision disorder or retinal dystrophy is selected from the group consisting of age-related macular degeneration, macular telangiectasia, retinitis pigmentosa, diabetic retinopathy, retinal vein occlusion, glaucoma, choroideremia, Sorsby fundus dystrophy, adult vitelliform macular dystrophy, Best's disease, Leber's congenital amaurosis, and X-linked retinoschisis. Preferably, the vision disorder is retinitis pigmentosa, age-related macular degeneration, or diabetic retinopathy.

[0122] In any embodiment, the subject has been diagnosed with a condition associated with or caused by the degeneration or loss of cone photoreceptor cells described herein. Preferably, the individual has been diagnosed with cone dystrophy. The individual may be diagnosed with progressive cone dystrophy or stationary cone dystrophy. The cone dystrophy may be rod-cone dystrophy or cone-rod dystrophy.

[0123] In some such embodiments, the method further comprises detecting a change in a condition or disorder symptom, including any symptom described herein. In some such embodiments, the change comprises a stabilization of the health of existing or reprogrammed cone cells and / or a decrease in the rate of vision loss in the subject. In certain such embodiments, the change comprises an improvement in the subject's vision.

[0124] In some such embodiments, the methods further include detecting a change in a condition or disorder symptom, where the change comprises an increase in the subject's ability to perceive color.

[0125] In any aspect of the present invention, the isolated nucleic acid of the present invention described herein, the AAV vector of the present invention described herein, the recombinant AAV of the present invention described herein, or the pharmaceutical composition of the present invention described herein is administered to a subject via the retina.In other words, the isolated nucleic acid of the present invention described herein, the AAV vector of the present invention described herein, the recombinant AAV of the present invention described herein, or the pharmaceutical composition of the present invention described herein is administered by retinal administration.Typically, the retinal administration is a retinal injection (e.g., intravitreal injection or subretinal injection) into the affected eye of the subject described above.

[0126] In another aspect, the invention provides a composition comprising any of the AAV vectors or rAAV of the invention disclosed herein and a pharma- ceutically acceptable carrier, excipient or diluent.

[0127] As used herein, unless the context otherwise requires, the term "comprise" and variations of terms such as "comprising", "comprises" and "comprised" are not intended to exclude additional additives, components, items or steps.

[0128] Further aspects of the invention and further embodiments of the aspects described in the preceding paragraphs will become apparent from the following description, given by way of example and with reference to the accompanying drawings, in which: [Brief description of the drawings]

[0129] [Figure 1] Experimental setup for genome-wide CRISPRa screening for genes that promote the reprogramming of human Müller glia (MG) cells into pyramidal cells. [Diagram 2] Genome-wide CRISPRa screening. A) Genome-wide CRISPRa screening of identified genes for iCone reprogramming, including the top hit NEUROG2. B) Gene ontology analysis showed that the candidate genes are related to photoreceptor signaling and function. [Diagram 3] The identified transcription factors form an important transcriptional network with NEUROG2 as a core factor (arrow). [Figure 4] Schematic diagram of in vitro reprogramming of human MG cells (MIOM1) into iCone. [Diagram 5] Characterization of photoreceptors by iCones reprogramming. A) iCones expressed L / M-opsin (OPN1LW / MW-DsRed+). B) Gene expression profiling showed that iCones upregulated cone marker genes. C) Multielectrode arrays showed that iCones had functional electrophysiology. [Figure 6] Initial screening of transcription factor cocktails for iCone reprogramming. Dotted lines indicate a 2-fold increase compared to control. Ng:NEUROG2, C:CRX, R:RAX, Roa:RORA, N:NEUROD1, O:OTX2, A:ASCL1, P:PAX6, T:THRB, M:MEF2C, F:FOXP1, On:ONECUT1. [Figure 7]A) Optimization of transcription factor cocktails for reprogramming human MG cells into iCone in a primary screen and subsequent B) validation in a secondary screen (n=3-4 biological replicates). C:CRX, M:MEF2C, T:THRB, R:RAX, N:NEUROD1, Roa:RORA, O:OTX2, P:PAX6, F:FOXP1, A:ASCL1, Ng:NEUROG2, On:ONECUT1. [Figure 8] A) Schematic of the in vivo reprogramming study in a rat retinitis pigmentosa (RP) model with photoreceptor degeneration (P23H3). Intravitreal injections of adeno-associated viruses (AAV) carrying iCone genes were performed in P23H3 rats and visual responses were analyzed using electroretinograms (ERG) 4 weeks after treatment. B) Schematic of the AAV vectors used to deliver individual iCone genes driven by the Müller glia (MG)-specific promoter GFAP and generated using the MG-specific targeting AAV serotype ShH10Y. [Figure 9] ERG analysis of P23H3 rats after injection of AAV carrying iCone genes Ascl1+Crx+Ng (ACNg), highlighting the functional improvement of visual responses after AAV delivery of iCone genes. Normalized a-wave (A,C), indicating photoreceptor function, and b-wave (B,D), indicating bipolar function, were performed before and after treatment of individual eyes. Naive controls and sham controls with PBS injection were used as negative controls. ***: p>0.001. [Figure 10] Immunohistochemistry analysis showed a focal increase in the thickness of the external nuclear layer (ONL) (indicated by white arrows) in P23H3 rats following treatment with AAV delivery of Ascl1+Crx+Neurog2 (ACNg) compared to untreated controls. DAPI was used as a nuclear counterstain along with the photoreceptor marker recoverin. INL: inner nuclear layer, ONL: outer nuclear layer.

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[0137] JPEG2025503495000008.jpg112145 DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0138] It will be understood that the invention disclosed and defined herein extends to all alternative combinations of two or more of the individual features mentioned or apparent from the text or drawings, all of these different combinations constituting various alternative aspects of the invention.

[0139] Reference will now be made in detail to specific embodiments of the invention. While the invention will be described in conjunction with the embodiments, it will be understood that the intention is not to limit the invention to those embodiments. On the contrary, the invention is intended to cover all alternatives, modifications, and equivalents which may be included within the scope of the present invention as defined by the claims.

[0140] Those skilled in the art will recognize many methods and materials similar or equivalent to those described herein that can be used to practice the present invention. The present invention is in no way limited to the methods and materials described. It will be understood that the invention disclosed and defined herein extends to all alternative combinations of two or more of the individual features mentioned or evident from the text or drawings. All of these different combinations constitute various alternative aspects of the invention.

[0141] For purposes of interpreting this specification, terms used in the singular will also include the plural and vice versa.

[0142] Currently, there are no suitable human cone photoreceptor cell lines available, which represents a bottleneck in the study of retinal diseases. It is therefore important to develop a good in vitro model for human cone photoreceptors for the retinal research field, as some questions regarding the intrinsic properties of the human visual system cannot be answered by animal models. The work described herein is the development of a direct reprogramming method to generate human cone photoreceptors in vitro. Importantly, the direct reprogramming method is much faster (approximately 2 weeks) compared to iPSC generation and differentiation (approximately 3-6 months), making it more cost-effective to generate human cone photoreceptors in vitro. The derived human cone photoreceptors provide a better in vitro model to study both retinal biology and disease, and provide a platform for drug testing in clinically relevant cell types, as well as a source of cells for transplantation for tissue engineering and cell therapy.

[0143] Furthermore, the invention described herein also includes in vivo reprogramming of cells into photoreceptor cells, directly demonstrating the application of in vivo gene therapy. In particular, we demonstrate the use of gene therapy approaches to prevent vision loss in a rat photoreceptor degeneration model. P23H is a well-established rat model for retinitis pigmentosa caused by rhodopsin mutations that undergoes gradual photoreceptor loss characteristic of human autosomal dominant retinitis pigmentosa. We performed viral delivery of a representative set of transcription factors by subretinal injection into P23H rats and analyzed visual function using electroretinograms (ERGs) before and after 4 weeks of treatment. The results shown in the Examples demonstrate that P23H rats treated with a representative set of factors prevented progressive loss after 4 weeks compared to untreated controls, providing direct supporting evidence for the therapeutic potential of using various transcription factors described herein to prevent vision loss in vivo.

[0144] In one aspect, the present invention provides compositions and methods for directly reprogramming or transdifferentiating source cells into target cells without the source cells becoming induced pluripotent stem cells (iPS) immediately prior to becoming the target cells. Compared to iPS cell technology, transdifferentiation is highly efficient and results in a very low risk of teratoma formation for downstream applications.

[0145] The process of reprogramming a cell changes the type of progeny that the cell can produce and includes transdifferentiation, which provides a cell that exhibits at least one characteristic of another somatic cell type.

[0146] The source cell can be any cell type described herein, including somatic cells or diseased somatic cells. The somatic cell can be an adult cell or a cell derived from an adult. The diseased cell can be a cell that exhibits one or more detectable characteristics of a disease or condition, for example, the diseased cell can be a cancer cell that exhibits one or more clinical or biochemical markers of cancer. Examples of source cells include glial cells, such as Müller glia (MG) cells, astrocytes, and microglial cells.

[0147] As used herein, the term "somatic cell" refers to any cell that forms the body of an organism, as opposed to a germ cell. In mammals, germ cells (also known as "gametes") are the sperm and eggs that fuse during fertilization to produce cells called zygotes, from which the entire mammalian embryo develops. All other cell types in a mammal's body, except for sperm and eggs, the cells from which they are made (gametes) and undifferentiated stem cells, are somatic cells, and the internal organs, skin, bone, blood, and connective tissues are all composed of somatic cells. In some embodiments, the somatic cell is a "non-embryonic somatic cell," which means a somatic cell that is not present in or obtained from an embryo and does not result from the in vitro propagation of such a cell. In some embodiments, the somatic cell is an "adult somatic cell," which means a cell that is present in or obtained from an organism other than an embryo or fetus, or that results from the in vitro propagation of such a cell. Somatic cells can be immortalized to provide an unlimited supply of cells, for example, by increasing the levels of telomerase reverse transcriptase (TERT). For example, the level of TERT can be increased by increasing transcription of TERT from an endogenous gene or by introducing a transgene via any gene delivery method or system.

[0148] Unless otherwise indicated, the methods for reprogramming somatic cells can be performed in vitro or in vivo, with in vitro being performed using isolated somatic cells maintained in culture.

[0149] Suitable somatic cells are receptive to uptake of transcription factors, including genetic material encoding the transcription factors, or can be made receptive using methods commonly known in the scientific literature. Methods of enhancing uptake may vary depending on the cell type and expression system. Exemplary conditions used to prepare receptive somatic cells with suitable transduction efficiency are well known to those of skill in the art.

[0150] "Cone cells", also referred to herein as "cone photoreceptors" or "cones", refer to a subtype of photoreceptor cells in the retina of the eye that function best in relatively bright light. Cones are sensitive to certain wavelengths of light, thus aiding in color detection. In addition, cones respond more quickly to stimuli than rod photoreceptors and detect finer details and more rapid changes in an image than rods, thus aiding in highly acute vision for activities where visual details are of primary importance, such as reading and driving. Cones are easily identifiable in cross-sections of the retina by the cone-like shape of their outer segments. Cones are also easily identifiable by their location in the retina, a pore-density of cones that reside in a 1.5 mm depression located in the center of the macula of the retina, called the "fovea" or "foveal hole".

[0151] As used herein, the term "isolated cell" refers to a cell that has been removed from the organism in which it was originally found or the progeny of such a cell. Optionally, the cell has been cultured in vitro, e.g., in the presence of other cells. Optionally, the cell is later introduced into a second organism, or reintroduced into the organism from which the cell (or a cell that is its progeny) was isolated.

[0152] As used herein, the term "isolated population" with respect to an isolated population of cells refers to a population of cells that has been removed and separated from a mixed or heterogeneous population of cells. In some embodiments, an isolated population is a substantially pure population of cells as compared to the heterogeneous population from which the cells are isolated or enriched.

[0153] The term "substantially pure" refers, with respect to a particular cell population, to a population of cells that is at least about 75%, preferably at least about 85%, more preferably at least about 90%, and most preferably at least about 95% pure, relative to the cells that make up the total cell population. Again, the term "substantially pure" or "essentially purified" refers, with respect to a population of target cells, to a population of cells that contains less than about 20%, more preferably less than about 15%, 10%, 8%, 7%, and most preferably less than about 5%, 4%, 3%, 2%, 1%, or less than 1% of cells that are not target cells or their progeny, as defined herein.

[0154] A source cell is determined to be transformed into a target cell or become a target-like cell by the method of the present invention when it shows at least one characteristic of a target cell type, i.e., a cone photoreceptor cell. For example, human Muller glia are identified as transformed into a cone photoreceptor-like cell when the cell shows at least one characteristic of a cone photoreceptor cell type. Typically, the cell displays 1, 2, 3, 4, 5, 6, 7, 8 or more characteristics (or markers) of a cone photoreceptor cell. For example, if the target cell is a cone photoreceptor cell, the cell is identified or determined to be a cone photoreceptor-like cell when the upregulation or presence of any one or more photoreceptor cell markers and / or changes in cell morphology (preferably an increase in opsin mRNA expression) are detectable. Other examples of photoreceptor markers include ARR3, CNGB3, GNAT2, GNGT2, GRK7, GUCA1C, PDE6C, PDE6H, RXRG, THRB, OPN1LW, OPN1MW and OPN1SW, electrophysiological response in photopic conditions, for example, as described in the Examples. Additional examples of cone photoreceptor markers include opsins, which are light-detecting molecules. For example, red / green opsin (cone receptor cells), blue opsin (cone receptor cells), and recoverin (rod photoreceptor cells, cone receptor cells). In any aspect of the invention, the target cell characteristics can be determined by analysis of cell morphology, gene expression profile, activity assay, protein expression profile, surface marker profile, or differentiation potential. Examples of characteristics or markers include those described herein and known to those of skill in the art.

[0155] The transcription factors referred to herein are designated by the HUGO Gene Nomenclature Committee (HGNC) Symbol. Exemplary nucleotide sequences for each transcription factor are shown in Table 1 below. The nucleotide sequences are derived from the Ensembl database (Flicek et al. (2014). Nucleic Acids Research Volume 42, Issue D1.D749-D755) version 83. Any homologs, orthologs, or paralogs of the transcription factors referred to herein are also contemplated for use herein.

[0156] One of skill in the art will understand that this information can be used in practicing the methods of the invention, for example, for the purpose of providing increased amounts of a transcription factor to a source cell, or for the purpose of providing a source cell with a nucleic acid for recombinantly expressing a transcription factor, etc.

[0157] [Table 1]

[0158] The term "variant" refers to a polypeptide that is at least 70%, 80%, 85%, 90%, 95%, 98%, or 99% identical to the full-length polypeptide. The present invention contemplates the use of variants of the transcription factors described herein, including the sequences listed in Table 1. A variant can be a fragment of a full-length polypeptide or a naturally occurring splice variant. A variant can be a polypeptide that is at least 70%, 80%, 85%, 90%, 95%, 98%, or 99% identical to a fragment of a polypeptide, where the fragment is at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 98%, or 99%, so long as the full-length wild-type polypeptide or a domain thereof has a desired functional activity, such as the ability to promote conversion of a source cell type to a target cell type. In some embodiments, the domain is at least 100, 200, 300, or 400 amino acids in length, beginning at any amino acid position within the sequence and extending toward the C-terminus. Modifications known in the art to eliminate or substantially reduce the activity of a protein are preferably avoided. In some embodiments, the variant lacks the N-terminal and / or C-terminal portions of the full-length polypeptide, e.g., up to 10, 20, or 50 amino acids from either end. In some embodiments, the polypeptide has the sequence of a mature (full-length) polypeptide, meaning a polypeptide that had one or more portions, such as a signal peptide, removed during normal intracellular proteolytic processing (e.g., during co-translational or post-translational processing). In some embodiments, where the protein is produced by other than purification from cells that naturally express it, the protein is a chimeric polypeptide, meaning that the protein contains portions from two or more different species. In some embodiments, where the protein is produced by other than purification from cells that naturally express it, the protein is a derivative, meaning that the protein contains additional sequences not associated with the protein, so long as they do not substantially reduce the biological activity of the protein.A person skilled in the art will know or can easily confirm whether a particular polypeptide variant, fragment, or derivative is functional using assays known in the art. For example, the ability of a transcription factor variant to convert a source cell into a target cell type can be assessed using the assays disclosed herein in the Examples. Other convenient assays include measuring the ability to activate transcription of a reporter construct that includes a transcription factor binding site operably linked to a nucleic acid sequence encoding a detectable marker, such as luciferase. In certain embodiments of the invention, a functional variant or fragment has at least 50%, 60%, 70%, 80%, 90%, 95% or more of the activity of the full-length wild-type polypeptide.

[0159] As used herein, the terms "biological activity" and "biologically active" refer to activity attributable to a particular biological element in a cell. For example, the biological activity of a polypeptide or a functional fragment or variant thereof refers to the ability of the polypeptide or a functional fragment or variant thereof to perform its native function, such as, for example, binding, enzymatic activity, etc. For example, a biologically active fragment or variant of a transcription factor retains the ability to bind to DNA and regulate transcription. Typically, the biologically active fragment or variant thereof regulates transcription to at least 70%, 75%, 80%, 85%, 90% or 95% of the level of the wild-type protein, preferably human.

[0160] Furthermore, the biological activity of a gene regulatory element, e.g., a promoter, enhancer, Kozak sequence, and the like, refers to the ability of the regulatory element or a functional fragment or variant thereof to translationally regulate, i.e., promote, enhance, or activate, respectively, the expression of a gene to which it is operably linked.

[0161] With respect to increasing the amount of a transcription factor, the term "increasing the amount of" refers to increasing the amount of the transcription factor in a cell of interest (e.g., a source cell such as a fibroblast or keratinocyte cell). In some embodiments, the amount of a transcription factor is "increased" in a cell of interest (e.g., a cell into which an expression cassette directing the expression of one or more transcription factor-encoding polynucleotides has been introduced) when the amount of the transcription factor is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more compared to a control (e.g., a glial cell into which none of the above-mentioned expression cassettes have been introduced). However, any method of increasing the amount of a transcription factor is contemplated, including any method of increasing the amount, rate, or efficiency of transcription, translation, stability, or activity of a transcription factor (or a pre-mRNA or mRNA encoding it).

[0162] In a particularly preferred embodiment, the method may include the use of the CRISPR activation system (CRISPRa) or variations thereof to activate / increase expression of endogenous genes in the source cells encoding transcription factors of which increased amounts are desired to facilitate reprogramming. Such methods are well known to those of skill in the art, such as those published in Fang et al. Molecular therapy. Nucleic Acids, 20 Nov 2018, 14:184-191 (herein incorporated by reference).

[0163] Additionally, downregulation or interference with negative regulators of transcriptional expression, increasing the efficiency of existing transcription (eg, SINEUP) is also contemplated.

[0164] The term "agent" as used herein refers to any compound or substance, such as, but not limited to, a small molecule, a nucleic acid, a polypeptide, a peptide, a drug, an ion, etc. An "agent" can be any chemical entity or moiety, including, but not limited to, synthetic and naturally occurring proteinaceous and non-proteinaceous entities. In some embodiments, the agent is a nucleic acid, a nucleic acid analog, a protein, an antibody, a peptide, an aptamer, an oligomer of nucleic acid, an amino acid, or a carbohydrate, including, but not limited to, a protein, an oligonucleotide, a ribozyme, a DNA enzyme, a glycoprotein, an siRNA, a lipoprotein, an aptamer, and modifications and combinations thereof. For example, a system or set of components, such as the CRISPR activation system, as described herein, is also contemplated as an agent.

[0165] The term "exogenous" when used with respect to a protein, gene, nucleic acid, or polynucleotide in a cell or organism refers to a protein, gene, nucleic acid, or polynucleotide that has been introduced into the cell or organism by artificial or natural means, or with respect to a cell, refers to a cell that has been isolated and then introduced into another cell or organism by artificial or natural means. An exogenous nucleic acid may be from a different organism or cell, or it may be one or more additional copies of a nucleic acid that occurs naturally in the organism or cell. A foreign cell may be from a different organism or from the same organism. As a non-limiting example, an exogenous nucleic acid is a nucleic acid that is in a different chromosomal location than in a native cell, or that is otherwise adjacent to a nucleic acid sequence that is different from the nucleic acid sequence found in nature. An exogenous nucleic acid may also be extrachromosomal, such as an episomal vector.

[0166] The methods of the present invention include high throughput screening applications. For example, high throughput screening assays may be used, including any of the assays according to the present invention, in which an aliquot of a system allowing for the production or expression of a transcription factor is exposed to multiple candidate agents in different wells of a multi-well plate. Furthermore, high throughput screening assays according to the present disclosure include aliquots of a system allowing for the production or expression of a transcription factor exposed to multiple candidate agents in any type of miniaturized assay system.

[0167] The methods of the present disclosure may be "miniaturized" in an assay system through any acceptable miniaturization method, including but not limited to multi-well plates, such as 24, 48, 96, or 384 wells per plate, microchip, or slide. Assays may be reduced in size to be performed on a microchip support, advantageously involving smaller amounts of reagents and other materials. Any miniaturization of the process conducive to high throughput screening is within the scope of the present invention.

[0168] In any of the methods of the present invention, the target cells can be transferred to the same mammal from which the source cells were obtained. In other words, the source cells used in the methods of the present invention can be autologous cells, i.e., can be obtained from the same individual to which the target cells are administered. Alternatively, the target cells can be allogeneically transferred to another individual. Preferably, the cells are autologous to the subject in the method of treating or preventing a medical condition in the individual.

[0169] As used herein, "culturing" refers to contacting cells with cell culture medium for a time and under conditions sufficient to allow, for example, differentiation or proliferation of the cells. The term "cell culture medium" (also referred to herein as "culture medium" or "medium") referred to herein is a medium for culturing cells that contains nutrients that maintain cell viability and support proliferation. Cell culture medium may contain other components such as salts, buffers, amino acids, glucose or other sugars, antibiotics, serum or serum replacement, and peptide growth factors in any of the following suitable combinations. Cell culture media commonly used for specific cell types are known to those of skill in the art. Exemplary cell culture media for use in the methods of the present invention are shown in Table 2.

[0170] [Table 2]

[0171] The nucleic acids described herein, or vectors containing the nucleic acids, can include one or more of the sequences set forth above in Table 1, or a sequence encoding any one or more of the amino acid sequences listed in Table 1.

[0172] The term "expression" refers to the cellular processes involved in the production of RNA and proteins, and, where applicable, secreted proteins, including, but not limited to, transcription, translation, folding, modification and processing.

[0173] The terms "isolated" or "partially purified" as used herein refer to a nucleic acid or polypeptide that has been separated from at least one other component (e.g., a nucleic acid or polypeptide) that is present with the nucleic acid or polypeptide as found in its natural source and / or that is present with the nucleic acid or polypeptide when expressed by a cell or secreted, in the case of a secreted polypeptide. A nucleic acid or polypeptide that is chemically synthesized or synthesized using in vitro transcription / translation is considered "isolated."

[0174] The term "vector" refers to a carrier DNA molecule into which a DNA sequence can be inserted for introduction into a host or source cell. Preferred vectors are those capable of autonomous replication and / or expression of the nucleic acid to which they are linked. Vectors capable of directing the expression of a gene to which they are operably linked are referred to herein as "expression vectors". Thus, an "expression vector" is a specialized vector that contains the necessary regulatory regions required for the expression of a gene of interest in a host cell. In some embodiments, the gene of interest is operably linked to another sequence within the vector. The vector can be a viral vector or a non-viral vector. When using a viral vector, it is preferred that the viral vector is replication-defective, which can be achieved, for example, by removing all viral nucleic acid that codes for replication. Replication-defective viral vectors still retain their infectious properties and enter cells in a similar manner to replicating adenoviral vectors, but once the replication-defective viral vector enters a cell, the replication-defective viral vector does not replicate or grow. Vectors also encompass liposomes and nanoparticles, and other means of delivering DNA molecules to cells.

[0175] The term "AAV" is an abbreviation for adeno-associated virus and may be used to refer to the virus itself or its derivatives. The term encompasses all subtypes, and both naturally occurring and recombinant forms, unless otherwise required. The term "AAV" includes AAV type 1 (AAV-1), AAV type 2 (AAV-2), AAV type 3 (AAV-3), AAV type 4 (AAV-4), AAV type 5 (AAV-5), AAV type 6 (AAV-6), AAV type 7 (AAV-7), AAV type 8 (AAV-8), avian AAV, bovine AAV, canine AAV, feline AAV, primate AAV, non-primate AAV, and ovine AAV. "Primate AAV" refers to AAV that infects primates, "non-primate AAV" refers to AAV that infects non-primate mammals, "bovine AAV" refers to AAV that infects bovine mammals, and so on.

[0176] "AAV virus" or "AAV virus particle" or "rAAV vector particle" refers to a viral particle composed of at least one AAV capsid protein (typically all of the capsid proteins of wild-type AAV) and an encapsidated polynucleotide rAAV vector. When the particle contains a heterologous polynucleotide (i.e., a polynucleotide other than the wild-type AAV genome, such as a transgene delivered to a mammalian cell), it is typically referred to as an "rAAV vector particle" or simply an "rAAV vector". Thus, since such a vector is contained in the rAAV particle, the production of the rAAV particle necessarily includes the production of the rAAV vector.

[0177] The term "replication defective" as used herein with respect to the AAV viral vector of the present invention means that the AAV vector cannot independently replicate and package its genome. For example, when a subject's cell is infected with rAAV virion, the heterologous gene is expressed in the infected cell, but the rAAV cannot further replicate due to the fact that the infected cell lacks the AAV rep and cap genes and accessory function genes.

[0178] As used herein, an "AAV variant" or "AAV mutant" refers to a viral particle that is composed of: a) a variant AAV capsid protein, where the variant AAV capsid protein contains at least one amino acid difference (e.g., an amino acid substitution, an amino acid insertion, an amino acid deletion) compared to a corresponding parent AAV capsid protein, and where the variant capsid protein confers increased infectivity of retinal cells compared to infectivity of retinal cells by AAV virions containing the corresponding parent AAV capsid protein, and where the AAV capsid protein does not contain an amino acid sequence present in a naturally occurring AAV capsid protein; and b) a heterologous nucleic acid that comprises a nucleotide sequence encoding a heterologous gene product.

[0179] The abbreviation "rAAV" refers to recombinant adeno-associated virus, also referred to as recombinant AAV vector (or "rAAV vector"). As used herein, "rAAV vector" refers to an AAV vector that contains a polynucleotide sequence that is not of AAV origin (i.e., a polynucleotide that is heterologous to AAV), typically a sequence of interest for genetic transformation of a cell, such as a transgene as described herein. Generally, the heterologous polynucleotide is flanked by at least one, and generally two, AAV inverted terminal repeats (ITRs). The term rAAV vector encompasses both rAAV vector particles and rAAV vector plasmids.

[0180] As used herein, a transgene is a gene (e.g., DNA or RNA, preferably mRNA) that is delivered to a cell by a vector.

[0181] The term "operably linked" means that regulatory sequences required for expression of a coding sequence are positioned on a DNA molecule in an appropriate position relative to the coding sequence to affect expression of the coding sequence. This same definition is sometimes applied to the placement of coding sequences and transcription control elements (e.g., promoters, enhancers, and termination elements) in an expression vector. The term "operably linked" includes having an appropriate initiation signal (e.g., ATG) in front of the polynucleotide sequence to be expressed and maintaining the correct reading frame to permit expression of the polynucleotide sequence under the control of the expression control sequences and production of the desired polypeptide encoded by the polynucleotide sequence.

[0182] The term "viral vector" refers to the use of a virus or virus-associated vector as a carrier of a nucleic acid construct into a cell. The construct may incorporate and package a non-replication defective viral genome, such as adenovirus, adeno-associated virus (AAV), or herpes simplex virus (HSV), including retroviral and lentiviral vectors, for infection and transduction of a cell. The vector may or may not integrate into the genome of the cell. The construct may include viral sequences for transfection, if desired. Alternatively, the construct may be incorporated into a vector capable of episomal replication, such as EPV and EBV vectors.

[0183] As used herein, the term "adenovirus" refers to viruses of the Adenovirida family. Adenoviruses are medium-sized (90-100 nm), non-enveloped (naked), icosahedral viruses composed of a nucleocapsid and a double-stranded linear DNA genome.

[0184] As used herein, the term "non-integrating viral vector" refers to a viral vector that does not integrate into the host genome, and the expression of genes delivered by the viral vector is transient. Since there is little or no integration into the host genome, non-integrating viral vectors have the advantage of not producing DNA mutations by inserting at random points in the genome. For example, non-integrating viral vectors remain extrachromosomal and do not insert their genes into the host genome, potentially inhibiting the expression of endogenous genes. Non-integrating viral vectors can include, but are not limited to, adenoviruses, alphaviruses, picornaviruses, and vaccinia viruses. These viral vectors are "non-integrating" viral vectors as that term is used herein, even though any of them may, in some rare circumstances, integrate viral nucleic acid into the genome of a host cell. What is important is that the viral vectors used in the methods described herein do not integrate their nucleic acid into the genome of a host cell as a rule or for the majority of their lifespan under the conditions used.

[0185] The vectors described herein can be constructed and engineered using methods commonly known in the scientific literature to enhance the safety of their use in therapy, to include selection and enrichment markers as necessary, and to optimize the expression of the nucleotide sequences contained therein. The vector should include structural components that allow the vector to self-replicate in the source cell type. For example, the known Epstein Barr oriP / Nuclear Antigen-1 (EBNA-I) combination (see, for example, Lindner, SE and B. Sugden, The plasmid replicon of Epstein-Barr virus: mechanistic insights into efficient, licensed, extrachromosomal replication in human cells, Plasmid 58:1 (2007), incorporated by reference herein as if set forth in its entirety) is sufficient to support the self-replicating vector and other combinations known to function in mammalian, particularly primate, cells can also be used. Standard techniques for the construction of expression vectors suitable for use in the present invention are well known to those of skill in the art and can be found in publications such as Sambrook J, et al., "Molecular cloning: a laboratory manual," (3rd ed. Cold Spring harbor Press, Cold Spring Harbor, NY 2001), which is incorporated by reference herein as if set forth in its entirety.

[0186] In the methods of the present invention, genetic material encoding the relevant transcription factors required for conversion is delivered to the source cell via one or more reprogramming vectors. Each transcription factor can be introduced into the source cell as a polynucleotide transgene encoding the transcription factor operably linked to a heterologous promoter capable of driving expression of the polynucleotide in the source cell.

[0187] Suitable reprogramming vectors are any of those described herein, including episomal vectors such as plasmids that do not encode all or part of a viral genome sufficient to produce an infectious or replication-competent virus, although the vector may contain structural elements derived from one or more viruses. One or more reprogramming vectors can be introduced into a single source cell. One or more transgenes can be provided on a single reprogramming vector. One strong constitutive transcription promoter can provide transcriptional control of multiple transgenes that can be provided as expression cassettes. Separate expression cassettes on a vector may be under transcriptional control of separate strong constitutive promoters, which may be copies of the same promoter or may be separate promoters. A variety of heterologous promoters are known in the art and may be used depending on factors such as the desired expression level of the transcription factor. As exemplified below, it may be advantageous to use different promoters with different strengths in the source cell to control the transcription of separate expression cassettes. Another consideration in the selection of transcription promoters is the rate at which the promoter is silenced. Those skilled in the art will understand that it may be advantageous to reduce the expression of one or more transgenes or transgene expression cassettes after the product of the gene has completed or substantially completed its role in the reprogramming method. Exemplary promoters are the human EF1α elongation factor promoter, the CMV cytomegalovirus immediate early promoter and the CAG chicken albumin promoter, as well as corresponding homologous promoters from other species. In human somatic cells, both EF1α and CMV are strong promoters, but the CMV promoter is silenced more efficiently than the EF1α promoter, such that the expression of the transgene under the control of the CMV promoter is turned off earlier than the expression of the transgene under the control of the EF1α promoter. Transcription factors can be expressed in source cells in relative ratios that can be varied to modulate reprogramming efficiency.Preferably, when multiple transgenes are encoded on a single transcript, an internal ribosome entry site is provided upstream of the transgene, distal from the transcription promoter. The relative ratios of factors may vary depending on the factors being delivered, but the skilled artisan in possession of this disclosure will be able to determine the optimal ratios of factors.

[0188] Those skilled in the art will understand that the advantageous efficiency of introducing all factors via a single vector rather than via multiple vectors becomes increasingly difficult to introduce as the total vector size increases. Those skilled in the art will also understand that the location of the transcription factor on the vector can affect its temporal expression and the resulting reprogramming efficiency. Thus, the applicant has employed various combinations of factors for vector combinations. Several such combinations are presented herein to support reprogramming.

[0189] After introduction of the reprogramming vector and while the source cell is being reprogrammed, the vector can persist in the target cell while the introduced transgene is transcribed and translated. Transgene expression can be advantageously downregulated or turned off in cells reprogrammed to the target cell type. The reprogramming vector can remain extrachromosomal. With very low efficiency, the vector can be integrated into the genome of the cell. The following examples are intended to illustrate the present invention but are in no way limiting.

[0190] Suitable methods for nucleic acid delivery for transformation of cells for use in the present invention are contemplated to include virtually any method by which a nucleic acid (e.g., DNA) can be introduced into a cell, as described herein or known to one of skill in the art (e.g., Stadtfeld and Hochedlinger, Nature Methods 6(5):329-330 (2009); Yusa et al., Nat. Methods 6:363-369 (2009); Woltjen, et al., Nature 458,766-770 (9 Apr. 2009)). Such methods include, but are not limited to, direct delivery of DNA, e.g., by ex vivo transfection (Wilson et al., Science,244:1344-1346, 1989; Nabel and Baltimore, Nature 326:711-713, 1987), optionally using lipid-based transfection reagents such as Fugene6 (Roche) or Lipofectamine (Invitrogen) by injection (U.S. Pat. Nos. 5,994,624, 5,981,274, 5,945,100, 5,780,448, 5,736,524, 5,702,932, 5,656,610, 5,589,466, and 5,580,859, each of which is incorporated herein by reference), including microinjection (Harland and Weintraub, J. Cell 2002, 14:131-132, 1987). Biol., 101:1094-1099, 1985; U.S. Pat. No. 5,789,215, incorporated herein by reference), by electroporation (U.S. Pat. No. 5,384,253, incorporated herein by reference; Tur-Kaspa et al., Mol. Cell Biol., 6:716-718, 1986; Potter et al., Proc. Nat'l Acad. Sci. USA, 81:7161-7165, 1984), by calcium phosphate precipitation (Graham and Van Der Eb, Virology, 52:456-467, 1973; Chen and Okayama, Mol. Cell Biol.,7(8):2745-2752,1987; Rippe et al.,Mol. Cell Biol.,10:689-695,1990), by using DEAE-dextran followed by polyethylene glycol (Gopal,Mol. Cell Biol.,5:1188-1190,1985), by direct sonic loading (Fechheimer et al.,Proc. Nat'l Acad. Sci. USA,84:8463-8467,1987), and by liposome-mediated transfection (Nicolau and Sene,Biochim. Biophys. Acta,721:185-190,1982; Fraley et al.,Proc. Nat'l Acad. Sci. USA,76:3348-3352,1979; Nicolau et al. al., Methods Enzymol., 149:157-176, 1987; Wong et al., Gene, 10:87-94, 1980; Kaneda et al., Science, 243:375-378, 1989; Kato et al., J. Biol. Chem., 266:3361-3364, 1991), and receptor-mediated transfection (Wu and Wu, Biochemistry, 27:887-892, 1988; Wu and Wu, J. Biol. Chem., 262:4429-4432, 1987), as well as any combination of such methods, each of which is incorporated herein by reference.

[0191] Several polypeptides capable of mediating the introduction of relevant molecules into cells have been previously described and can be adapted to the present invention, see, for example, Langel (2002) Cell Penetrating Peptides: Processes and Applications, CRC Press, Pharmacology and Toxicology Series. Examples of polypeptide sequences that enhance transport across membranes include, but are not limited to, the Drosophila homeoprotein Antennapedia transcription protein (AntHD) (Joliot et al., New Biol. 3:1121-34, 1991; Joliot et al., Proc. Natl. Acad. Sci. USA, 88: 1864-8, 1991; Le Roux et al., Proc. Natl. Acad. Sci. USA, 90:9120-4, 1993), herpes simplex virus structural protein VP22 (Elliott and O'Hare, Cell 88:223-33, 1997), the HIV-1 transcriptional activator TAT protein (Green and Loewenstein, Cell 55:1179-1188, 1988; Frankel and Pabo, Cell 55:1 289-1193, 1988), Kaposi's FGF signal sequence (kFGF), protein transduction domain-4 (PTD4), penetratin, M918, transportan-10, nuclear localization sequence, PEP-I peptide, amphipathic peptides (e.g., MPG peptide), delivery-enhancing transporters, such as those described in U.S. Pat. No. 6,730,293 (including, but not limited to, peptide sequences containing at least 5-25 or more consecutive arginines or 5-25 or more arginines in a contiguous set of 30, 40, or 50 amino acids, including, but not limited to, peptides having sufficient (e.g., at least 5) guanidino or amidino moieties), and the commercially available Penetratin™ 1 peptide, as well as Diatos Peptide Vectors ("DPVs") of the Vectocell® platform available from Daitos SA of Paris, France.See also WO / 2005 / 084158 and WO / 2007 / 123667, and the additional transporters described therein. Not only are these proteins able to cross the plasma membrane, but the connection of other proteins, such as the transcription factors described herein, is sufficient to stimulate cellular uptake of these complexes.

[0192] A "promoter" as used herein encompasses a DNA sequence that induces the binding of RNA polymerase, thereby facilitating RNA synthesis, i.e., a minimal sequence sufficient to direct transcription. The promoter and the expression of the corresponding protein or polypeptide can be ubiquitous, meaning that it is highly active in a wide variety of cells, tissues and species, or it can be cell type-specific (such as glial cell-specific), tissue-specific, or species-specific. The promoter can be "constitutive", meaning that it can be constantly active, or it can be "inducible", meaning that the promoter can be activated or inactivated by the presence or absence of a biotic or abiotic factor. The nucleic acid construct or vector of the present invention also includes enhancer sequences, which may or may not be contiguous with the promoter sequence. Enhancer sequences affect promoter-dependent gene expression and can be located in the 5' or 3' region of the native gene.

[0193] As used herein, "enhancer" includes cis-acting elements that stimulate or inhibit transcription of adjacent genes. Enhancers that inhibit transcription are also called "silencers." Enhancers can function (i.e., associate with coding sequences) in either orientation, over distances of up to several kilobase pairs (kb) from the coding sequence and downstream from the transcribed region.

[0194] As used herein, a "termination signal sequence" includes any genetic element that directs RNA polymerase to terminate transcription, such as a polyadenylation signal sequence.

[0195] As used herein, a "polyadenylation signal sequence" encompasses the recognition regions necessary for endonucleolytic cleavage of an RNA transcript, followed by the polyadenylation consensus sequence AATAAA. A polyadenylation signal sequence provides a "poly A site", i.e., a site on an RNA transcript at which adenine residues are added by post-transcriptional polyadenylation.

[0196] Gene Therapy Vectors Any convenient vector, such as gene therapy vector or gene delivery vector (used interchangeably herein) that is used to deliver nucleic acid or nucleotide sequence as described herein to cells in the retina, is included in the vector of the present disclosure.For example, the vector may comprise single-stranded or double-stranded nucleic acid, such as single-stranded or double-stranded DNA or RNA.For example, the gene delivery vector may be naked DNA or RNA, such as plasmid, minicircle, etc. As another example, the gene delivery vector may be a virus, such as an adenovirus, an adeno-associated virus (AAV), a baculovirus, or a retrovirus, such as Moloney murine leukemia virus (M-MuLV), Moloney murine sarcoma virus (MoMSV), Harvey murine sarcoma virus (HaMuSV), mouse mammary tumor virus (MuMTV), gibbon ape leukemia virus (GaLV), feline leukemia virus (FLV), spumavirus, Friend murine leukemia virus, murine stem cell virus (MSCV), and Rous sarcoma virus (RSV), or a lentivirus. Although embodiments involving the use of adeno-associated viruses are described in more detail below, it is expected that one of skill in the art will understand that similar knowledge and techniques in the art may be adapted to non-AAV gene therapy vectors. See, e.g., the discussion of retroviral vectors in U.S. Pat. Nos. 7,585,676 and 8,900,858, and the discussion of adenoviral vectors in, e.g., U.S. Pat. No. 7,858,367, the entire disclosures of which are incorporated herein by reference.

[0197] Gene therapy vectors, e.g., rAAV, lentivirus and baculovirus, virions encapsulating the polynucleotide cassettes of the present disclosure can be produced using standard methodologies. In some embodiments, the gene delivery vector is a recombinant adeno-associated virus (rAAV). In such embodiments, the expression constructs encoding the set of transcription factors described herein in (a)-(jj), or biologically active fragments or variants thereof, are flanked at the 5' and 3' ends by functional AAV inverted terminal repeat (ITR) sequences. By "functional AAV ITR sequences" is meant that the ITR sequences function as intended with respect to rescue, replication and packaging of AAV virions. Thus, AAV ITRs for use in the gene delivery vectors of the invention need not have wild-type nucleotide sequences, but may be altered by nucleotide insertions, deletions, or substitutions, or the AAV ITRs may be derived from any of several AAV serotypes, e.g., AAV1, AAV2, AAV3, AAV4, AAVS, AAV6, AAV7, AAV8, AAV9, AAV10, ShH10, and ShH10Y. Preferred AAV vectors have the wild-type REP and CAP genes deleted in whole or in part, but retain functional flanking ITR sequences.

[0198] In such an embodiment, the nucleic acid comprising the expression construct is encapsidated within an AAV capsid, which may be derived from any adeno-associated virus serotype, including, but not limited to, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, etc. For example, the AAV capsid may be a wild-type or naturally occurring capsid. Particularly interesting wild-type AAV capsids include AAV2, AAV5, and AAV9. However, similar to the ITRs, the capsid need not have a wild-type nucleotide sequence, but rather may be altered by insertion, deletion, or substitution of nucleotides in the VP1, VP2, or VP3 sequences, so long as the capsid is capable of transducing cone cells. In other words, the AAV capsid may be a variant AAV capsid. Variant AAV capsids of particular interest include those that contain a peptide insertion within residues 580-600 of AAV2, or the corresponding residues in another AAV, e.g., LGETTRP, NETITRP, KAGQANN, KDPKTTN, KDTDTTR, RAGGSVG, AVDTTKF, or STGKVPN, as disclosed in U.S. Application No. 2014 / 0294771, the entire disclosure of which is incorporated herein by reference. In some embodiments, the AAV vector is a "pseudotyped" AAV made by using the capsid (cap) gene of one AAV and the rep gene and ITRs from a different AAV, e.g., pseudotyped AAV2 made using rep from AAV2 and cap from AAV1, AAV3, AAV4, AAVS, AAV6, AAV7, AAV8, or AAV9, together with a plasmid containing an AAV2-based vector. For example, the AAV vector can be rAAV2 / 1, rAAV2 / 3, rAAV2 / 4, rAAV2 / 5, rAAV2 / 6, rAAV2 / 7, rAAV2 / 8, rAAV2 / 9, etc. Preferably, the rAAV is replication-deficient, in that the AAV vector is unable to further replicate and package its genome independently.For example, when cone cells are transduced with rAAV virions, genes are expressed in the transduced cone cells, but the rAAV cannot replicate due to the fact that the transduced cone cells lack the AAV rep and cap genes and accessory function genes.

[0199] In the case of rAAV virions, an AAV expression vector according to the invention may be introduced into a producer cell followed by an AAV helper construct, which comprises an AAV coding region capable of being expressed in the producer cell and which complements AAV helper functions in the absence of the AAV vector. This is followed by introduction of a helper virus and / or additional vectors into the producer cell, which provide accessory functions capable of supporting efficient rAAV virus production. The producer cells are then cultured to produce rAAV.

[0200] In preparing the rAAV composition, any host cell for producing rAAV virions can be used, including, for example, mammalian cells (e.g., 293 cells), insect cells (e.g., SF9 cells), microorganisms, and yeast. The host cell can also be a packaging cell in which the AAV rep and cap genes are stably maintained in the host cell, or a producer cell in which the AAV vector genome is stably maintained and packaged. Exemplary packaging and producer cells are derived from SF-9, 293, A549, or HeLa cells. The AAV vector is purified and formulated using standard techniques known in the art. These steps are performed using standard methodologies. Replication-defective AAV virions encapsulating the recombinant AAV vector of the invention are produced by standard techniques known in the art using AAV packaging cells and packaging techniques. Examples of these methods can be found, for example, in U.S. Patent Nos. 5,436,146, 5,753,500, 6,040,183, 6,093,570, and 6,548,286, which are expressly incorporated herein by reference in their entireties. Additional compositions and methods for packaging are described in Wang et al. (US 2002 / 0168342), which is also incorporated herein by reference in its entirety.

[0201] Any suitable method for producing viral particles for delivery of the nucleic acid (e.g., DNA or RNA) or nucleotide sequences described herein can be used, including, but not limited to, those described in the Examples below. Any concentration of viral particles suitable for efficiently transducing retinal cells can be prepared for contact with retinal cells in vitro or in vivo. For example, viral particles can be prepared at concentrations of 10 8 Vector genomes / mL or more, e.g., 5 x 10 8 Vector genomes / mL, 10 9 Vector genomes / mL, 5 × 10 9 Vector genomes / mL, 10 10 Vector genomes / mL, 5 × 10 10Vector genomes / mL, 10 11 Vector genomes / mL, 5 × 10 11 Vector genomes / mL, 10 12 Vector genomes / mL, 5 × 10 12 Vector genomes / mL, 10 13 Vector genomes / mL, 1.5 × 10 13 Vector genomes / mL, 3 × 10 13 Vector genomes / mL, 5 × 10 13 Vector genomes / mL, 7.5 × 10 13 Vector genomes / mL, 9 × 10 13 Vector genomes / mL, 1 × 10 14 Vector genomes / mL, 5 × 10 14 vector genomes / mL or more, but typically 1 x 10 15 The vector may be formulated at a concentration of up to 10 vector genomes / mL. Similarly, any total number of viral particles may be administered to a mammalian or primate eye suitable to provide adequate transduction of retinal cells to impart a desired effect or treat a disease. In various preferred embodiments, at least 10 viral particles per eye are administered. 8 , 5×10 8 , 10 9 , 5×10 9 , 10 10 , 5×10 10 , 10 11 , 5×10 11 , 10 12 , 10 13 , 5×10 12 , 10 13 , 1.5×10 13 , 3×10 13 , 5×10 13 , 7.5×10 13 , 9×10 13 , 1×10 14 virus particles, or 5 x 10 14 10 virus particles or more, but typically 1 x 10 15No more than 100 viral particles are injected. Any suitable number of administrations of the vector may be administered to the subject's eye. In one embodiment, the method includes a single administration, while in other embodiments, multiple administrations are administered over a period of time deemed appropriate by the attending clinician.

[0202] The vector may be, but is not limited to, 1×10 8 vector genomes or more, e.g., 1 x 10 9 , 1×10 10 , 1×10 11 , 1×10 12 , or 1 × 10 13 vector genomes, or more, in certain cases up to 1 x 10 14 vector genomes, but typically 4 × 10 15 The vector may be formulated into any suitable unit dosage containing up to about 5×10 vector genomes. In some cases, the unit dosage is up to about 5×10 15 vector genome, e.g., 1 x 10 14 vector genomes or less, e.g., 1 x 10 13 , 1×10 12 , 1×10 11 , 1×10 10 , or 1×10 9 vector genomes or less, in certain cases 1 x 10 8 vector genome or less, typically 1 x 10 8 In some cases, the unit dosage is 1×10 10 ~1×10 11 In some cases, the unit dosage is 1×10 10 ~3×10 12 In some cases, the unit dosage is 1×10 9 ~3×10 13 In some cases, the unit dosage is 1×10 8 ~3×10 14 The vector genome.

[0203] In some cases, the unit dosage of a pharmaceutical composition can be measured using the multiplicity of infection (MOI). MOI refers to the ratio, or multiplicity, of vector or viral genome to cells to which the nucleic acid can be delivered. In some cases, the MOI is 1×10 6 In some cases, the MOI can be 1×10 5 ~1×10 7 In some cases, the MOI can be 1×10 4 ~1×10 8 In some cases, the recombinant virus of the disclosure can be at least about 1 x 10 1 , 1×10 2 , 1×103, 1×10 4 , 1×10 5 , 1×10 6 , 1×10 7 , 1×10 8 , 1×10 9 , 1×10 10 , 1×10 11 , 1×10 12 , 1×10 13 , 1×10 14 , 1×10 15 , 1×10 16 , 1×10 17 , and 1 × 10 18 In some cases, the recombinant viruses of the disclosure are at an MOI of 1 x 10 8 ~3×10 14 In some cases, the recombinant viruses of the present disclosure are at an MOI of up to about 1 x 10 1 , 1×10 2 , 1×10 3 , 1×10 4 , 1×10 5 , 1×10 6 , 1×10 7 , 1×10 8 , 1×10 9 , 1×10 10 , 1×10 11 , 1×10 12 , 1×10 13 , 1×10 14 , 1×10 15 , 1×10 16 , 1×10 17 , and 1 × 1018 is the MOI.

[0204] In some embodiments, the amount of pharmaceutical composition is about 1×10 8 ~Approx. 1×10 15 of recombinant virus, approximately 1 × 10 9 ~Approx. 1×10 14 of recombinant virus, approximately 1 × 10 10 ~Approx. 1×10 13 of recombinant virus, or approximately 1 × 10 11 ~Approx. 3×10 12 The recombinant virus includes

[0205] formulation The nucleic acid or vector according to the present invention may be combined in a composition having several different forms, in particular depending on the method of using the composition. Thus, for example, the composition may be in the form of a powder, tablet, capsule, liquid, ointment, cream, gel, hydrogel, aerosol, spray, micellar solution, liposomal suspension, or any other suitable form that can be administered to a person or animal in need of treatment. However, preferably, the construct or vector is formulated for suitable administration to the subject's eye, preferably the retina, preferably by injection, more preferably by retinal injection (e.g. subretinal injection), or most preferably by intravitreal injection. It will be understood that the vehicle of the pharmaceutical according to the present invention should be well tolerated by the subject to which it is given.

[0206] It will be understood that the amount of nucleic acid or vector required is determined by its biological activity and bioavailability, which depends on the mode of administration, the physicochemical properties of the nucleic acid or vector, and whether it is used as a monotherapy or in a combination therapy. The frequency of administration is also influenced by the half-life of the construct or vector in the subject being treated. The optimal dosage to be administered may be determined by one skilled in the art and may vary depending on the particular nucleic acid or vector being used, the strength of the pharmaceutical composition, the mode of administration, and the progression of the retinal disorder. Additional factors depending on the particular subject being treated, including the subject's age, weight, sex, diet, and time of administration, may give rise to the need to adjust the dosage.

[0207] Generally, a daily dose of 0.001 μg / kg to 10 mg / kg of body weight, or 0.01 μg / kg to 1 mg / kg of body weight, of the nucleic acid or vector according to the invention may be used to treat, ameliorate, or prevent retinal disorders, depending on the nucleic acid or vector used.

[0208] The nucleic acid or vector may be administered before, during or after the onset of cone cell damage. The daily dose may be given as a single administration (e.g., one injection per day). Alternatively, the nucleic acid or vector may require more than one administration during the day. By way of example, the nucleic acid or vector may be administered as two (or more depending on the severity of the retinal damage being treated) daily doses of 0.07 μg to 700 mg (i.e., assuming a body weight of 70 kg). The patient undergoing treatment may take a first dose upon awakening, then a second dose in the evening (in the case of a two-dose regime), or at intervals of 3 or 4 hours thereafter. Alternatively, a sustained release device may be used to provide the patient with an optimal dose of the nucleic acid or vector according to the invention without the need to administer repeated doses.

[0209] However, the pharmaceutical vehicle may be liquid and the pharmaceutical composition is in the form of a solution. Liquid vehicles are used in the preparation of solutions, suspensions, emulsions, syrups, elixirs, and pressurized compositions. The nucleic acid or vector according to the present invention may be dissolved or suspended in a pharma- ceutical acceptable liquid vehicle, such as water, an organic solvent, a mixture of both, or a pharma- ceutically acceptable oil or fat.

[0210] Liquid pharmaceutical compositions that are sterile solutions or suspensions can be utilized, for example, by intraocular, particularly intravitreal or subretinal injection. The nucleic acid or vector can be prepared as a sterile solid composition that can be dissolved or suspended at the time of administration using sterile water, saline, or other appropriate sterile injectable medium.

[0211] When contacting retinal glial cells in vivo, the nucleic acid (e.g., synthetic mRNA) of the present invention described herein, or vector, preferably the AAV vector of the present invention described herein, or the recombinant AAV of the present invention described herein, can be processed to be suitable for delivery to the eye. In particular, the present invention includes pharmaceutical compositions comprising the nucleic acid of the present invention described herein, vector, preferably the AAV vector of the present invention described herein, or the recombinant AAV of the present invention described herein, and a pharma- ceutically acceptable carrier, diluent or excipient. The nucleic acid of the present invention described herein, vector, preferably the AAV vector of the present invention described herein, or the recombinant AAV of the present invention described herein, can generally be combined with pharma- ceutically acceptable carriers, diluents, and reagents useful in the preparation of safe, non-toxic, and desirable formulations, including excipients that are acceptable for use in primates. Such excipients can be solid, liquid, semi-solid, or, in the case of an aerosol composition, gaseous. Examples of such carriers or diluents include, but are not limited to, water, saline, Ringer's solution, dextrose solution, and 5% human serum albumin. Supplementary active compounds can also be incorporated into the formulation. The solution or suspension used in the formulation can include sterile diluents such as water for injection, saline solution, non-volatile oils, polyethylene glycol, glycerin, propylene glycol or other synthetic solvents; antibacterial compounds such as benzyl alcohol or methylparaben; antioxidants such as ascorbic acid or sodium hydrogen sulfate; chelating compounds such as ethylenediaminetetraacetic acid (EDTA); buffers such as acetate, citrate or phosphate; detergents such as Tween 20 to prevent aggregation; and compounds for adjusting osmotic pressure such as sodium chloride or dextrose. pH can be adjusted using acids or bases such as hydrochloric acid or sodium hydroxide.

[0212] Pharmaceutical compositions suitable for internal use in the present invention further include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, or phosphate buffered saline (PBS). In some cases, the composition should be sterile and fluid to the extent that easy syringability exists. In certain embodiments, the composition is stable under the conditions of manufacture and storage and is preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by maintaining the required particle size in the case of dispersions, and by the use of surfactants. Prevention of microbial action can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it is preferable to include isotonic agents, such as sugars, polyalcohols (such as mannitol, sorbitol, etc.), and sodium chloride in the composition. Prolonged absorption of the internal composition can be brought about by including in the composition an agent that delays absorption, such as aluminum monostearate and gelatin.

[0213] Sterile solutions can be prepared by incorporating the nucleic acid, vector, preferably the AAV vector of the present invention described herein, or the recombinant AAV of the present invention described herein, in the required amount into a suitable solvent with one or a combination of the ingredients listed above, as needed, followed by sterilization by filtration. In general, dispersions are prepared by incorporating the nucleic acid, vector, preferably the AAV vector of the present invention described herein, or the recombinant AAV of the present invention described herein, into a sterile vehicle containing a basic dispersion medium and other necessary ingredients listed above. In the case of sterile powders for preparing sterile injections, the preparation method is high vacuum drying and lyophilization, which obtains a powder of the active ingredient and any additional desired ingredients from the solution previously sterile-filtered.

[0214] In one embodiment, the active compound is prepared with a carrier that protects the compound from rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable and biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used. Methods for preparing such formulations will be clear to those skilled in the art. Materials can also be obtained commercially. Liposomal suspensions (including liposomes targeted to infected cells using monoclonal antibodies against viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Pat. No. 4,522,811.

[0215] The pharmaceutical compositions may be included in a container, pack, or dispenser, e.g., a syringe, e.g., a prefilled syringe, together with instructions for administration.

[0216] The pharmaceutical compositions of the present invention include any pharma- ceutically acceptable salts, esters, or salts of such esters, or any other compounds that can yield (directly or indirectly) biologically active metabolites or residues thereof upon administration to an animal, including a human. Thus, for example, the present disclosure is also directed to prodrugs and pharma- ceutically acceptable salts of the compounds of the present invention, pharma- ceutically acceptable salts of such prodrugs, and other bioequivalents.

[0217] The term "pharmaceutical acceptable salt" refers to a physiologically and pharma-ceutical acceptable salt of the compound of the present invention, i.e., a salt that retains the desired biological activity of the parent compound and does not impart undesired toxicological effects thereto.Various pharma-ceutical acceptable salts are known in the art and are described, for example, in "Remington's Pharmaceutical Sciences", 17th edition, Alfonso R. Gennaro (Ed.), Mark Publishing Company, Easton, Pa., USA, 1985 (and its more recent editions), in "Encyclopaedia of Pharmaceutical Technology", 3rd edition, James Swarbrick (Ed.), Informa Healthcare USA (Inc.), NY, USA, 2007, and in J.Pharm.Sci.66:2 (1977).For a review of suitable salts, see also Handbook of Pharmaceutical Salts: Properties, Selection, and Use by Stahl and Wermuth (Wiley-VCH, 2002).

[0218] Pharmaceutically acceptable base addition salts are formed with metals or amines, such as alkali and alkaline earth metals or organic amines. Metals used as cations include sodium, potassium, magnesium, calcium, and the like. Amines include N-N'-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, dicyclohexylamine, ethylenediamine, N-methylglucamine, and procaine (see, for example, Berge et al., "Pharmaceutical Salts," J. Pharma Sci., 1977, 66, 119). The base addition salts of the above-mentioned acidic compounds are prepared by contacting the free acid form with a sufficient amount of the desired base to produce the salt in a conventional manner. The free acid form may be regenerated by contacting the salt form with an acid and isolating the free acid in a conventional manner. The free acid forms differ slightly from their respective salt forms in certain physical properties, such as solubility in polar solvents, but in other respects the salts are equivalent to their respective free acids for purposes of the present invention.

[0219] The nucleic acid, vector, preferably the AAV vector of the invention described herein, or the recombinant AAV of the invention described herein, can be incorporated into a pharmaceutical composition for administration to a mammalian patient, specifically a primate, more specifically a human. The nucleic acid, vector, preferably the AAV vector of the invention described herein, or the recombinant AAV of the invention described herein, can be formulated in a non-toxic, inert, pharma- ceutically acceptable aqueous carrier, preferably at a pH in the range of 3-8, more preferably at a pH in the range of 6-8. Such a sterile composition comprises a vector or virion containing a nucleic acid encoding PHGDH or a biologically active fragment or variant thereof dissolved in an aqueous buffer having an acceptable pH upon reconstitution.

[0220] In some embodiments, the pharmaceutical compositions provided herein comprise a therapeutically effective amount of vectors or virions in admixture with pharma- ceutically acceptable carriers and / or excipients, such as saline, phosphate buffered saline, phosphates, and amino acids, polymers, polyols, sugars, buffers, preservatives, and other proteins. Exemplary amino acids, polymers, sugars, and the like are octylphenoxypolyethoxyethanol compounds, polyethylene glycol monostearate compounds, polyoxyethylene sorbitan fatty acid esters, sucrose, fructose, dextrose, maltose, glucose, mannitol, dextran, sorbitol, inositol, galactitol, xylitol, lactose, trehalose, bovine or human serum albumin, citrate, acetate, Ringer's solution and Hank's solution, cysteine, arginine, carnitine, alanine, glycine, lysine, valine, leucine, polyvinylpyrrolidone, polyethylene, and glycols. Preferably, the formulation is stable at 4° C. for at least 6 months.

[0221] In some embodiments, the pharmaceutical compositions provided herein contain a buffer, such as phosphate buffered saline (PBS) or sodium phosphate / sodium sulfate, Tris buffer, glycine buffer, sterile water, and other buffers known to those of skill in the art, such as those described in Good et al. (1966) Biochemistry 5: 467. The pH of the buffer in which the pharmaceutical composition containing the tumor suppressor gene contained in the adenoviral vector delivery system can be in the range of 6.5 to 7.75, preferably 7 to 7.5, and most preferably 7.2 to 7.4.

[0222] promoter Any promoter sequence that allows expression in the target tissue / cell type glial cells (preferably retinal glial cells) is useful in the present invention. These include ubiquitous promoters, such as CAG promoters, and glial cell-specific promoters. CAG promoters preferably include cytomegalovirus (CMV) early enhancer element, promoter, first exon and first intron of chicken beta-actin (CBA) gene, and splice acceptor of rabbit beta-globin gene. Examples of glial cell-specific promoters include, but are not limited to, the promoters of GFAP, GLAST, and RLBP1.

[0223] Treatment methods The invention provides a method of treating a condition associated with or caused by the degeneration or loss of photoreceptor cells in an individual in need thereof, the method comprising administering to the individual a cell or population of cells generated in vitro or ex vivo by any of the methods described herein.

[0224] The invention provides for the use of a cell or population of cells generated in vitro or ex vivo by any of the methods described herein in the manufacture of a medicament for the treatment of a condition associated with or caused by photoreceptor cell degeneration in an individual in need thereof.

[0225] The invention provides cells or cell populations generated in vitro or ex vivo by any of the methods described herein for use in an individual in need of treatment for a condition associated with or caused by photoreceptor cell degeneration.

[0226] In another aspect, the invention provides a method of reducing the progression of or reversing vision loss associated with or caused by the degeneration or loss of cone photoreceptor cells in a subject, the method comprising administering to the subject a nucleic acid of the invention as described herein, a vector of the invention as described herein, preferably an AAV vector, a recombinant AAV of the invention as described herein, or a pharmaceutical composition of the invention as described herein, thereby reducing the progression of or reversing vision loss associated with or caused by the degeneration or loss of cone photoreceptor cells.

[0227] In another aspect, the present invention provides the use of a nucleic acid of the invention as described herein, a vector of the invention as described herein, preferably an AAV vector, a recombinant AAV of the invention as described herein, or a pharmaceutical composition of the invention as described herein, in the manufacture of a medicament for reducing the progression of or reversing vision loss associated with or caused by degeneration or loss of cone photoreceptor cells in a subject.

[0228] In another aspect, the invention provides a nucleic acid of the invention as described herein, a vector of the invention as described herein, preferably an AAV vector, a recombinant AAV of the invention as described herein, or a pharmaceutical composition of the invention as described herein for use in reducing the progression of or restoring vision associated with or caused by degeneration or loss of cone photoreceptor cells in a subject.

[0229] In any embodiment, preferably the subject is a human.

[0230] In any aspect or embodiment, a condition associated with or caused by the degeneration or loss of cone photoreceptor cells may also be referred to as a cone cell disorder, which is associated with or causes a change in vision, typically a decrease in vision.

[0231] In some embodiments, the cone cell disorder is a retinal degenerative disorder. In certain embodiments, the retinal degenerative disorder is selected from the group consisting of achromatopsia, blue-cone monochromacy, protanopia, deutanopia, and tritanopia. In some embodiments, the cone cell disorder is a macular dystrophy or a retinal dystrophy. The macular dystrophy may be selected from the group consisting of Stargardt macular dystrophy, cone dystrophy (including rod-cone dystrophy and cone-rod dystrophy), spinocerebellar ataxia type 7, and Bardet-Biedl syndrome-1. Preferably, the macular dystrophy is Stargardt macular dystrophy or a cone-rod dystrophy. In some embodiments, the cone cell disorder is a central macular vision disorder or a retinal dystrophy. In certain embodiments, the central macular vision disorder or retinal dystrophy is selected from the group consisting of age-related macular degeneration, macular telangiectasia, retinitis pigmentosa, diabetic retinopathy, retinal vein occlusion, glaucoma, choroideremia, Sorsby fundus dystrophy, adult vitelliform macular dystrophy, Best's disease, Leber's congenital amaurosis, and X-linked retinoschisis. Preferably, the vision disorder is retinitis pigmentosa, age-related macular degeneration, or diabetic retinopathy.

[0232] In any embodiment, the subject has been diagnosed with a condition associated with or caused by the degeneration or loss of cone photoreceptor cells described herein. Preferably, the individual has been diagnosed with cone dystrophy. The individual may be diagnosed with progressive cone dystrophy or stationary cone dystrophy. The cone dystrophy may be rod-cone dystrophy or cone-rod dystrophy.

[0233] In some such embodiments, the method further comprises detecting a change in a condition or disorder symptom, including any symptom described herein. In some such embodiments, the change comprises a stabilization of the health of existing or reprogrammed cone cells and / or a decrease in the rate of vision loss in the subject. In certain such embodiments, the change comprises an improvement in the subject's vision.

[0234] In some such embodiments, the methods further include detecting a change in a condition or disorder symptom, where the change comprises an increase in the subject's ability to perceive color.

[0235] In any aspect of the invention, the isolated nucleic acid of the invention described herein, the AAV vector of the invention described herein, the recombinant AAV of the invention described herein, or the pharmaceutical composition of the invention described herein is administered to the retina of the subject, preferably by retinal injection (e.g., subretinal injection or intravitreal injection) into the diseased eye of the subject as described above.

[0236] In another aspect, the invention provides a composition comprising any of the AAV vectors or rAAV of the invention disclosed herein and a pharma- ceutically acceptable carrier, excipient or diluent.

[0237] In some embodiments, the photoreceptor loss is a complete loss of cone photoreceptors. In some embodiments, the patient has a visual acuity of 20 / 60 or less, including 20 / 80 or less, 20 / 100 or less, 20 / 120 or less, 20 / 140 or less, 20 / 160 or less, 20 / 180 or less, 20 / 200 or less, 20 / 400 or less, 20 / 800 or less, or 20 / 1000 or less.

[0238] Administration of the cells or cell populations to an individual in need thereof to treat a condition associated with or caused by degeneration of cone photoreceptor cells may be by any method known in the art.

[0239] In the method of the present invention, the cells to be transplanted are transferred to the recipient in any physiologically acceptable vehicle, including an isotonic vehicle prepared under sufficiently sterile conditions for human administration. For general principles of pharmaceutical formulations, the reader is referred to Cell Therapy: Stem Cell Transplantation, Gene Therapy, and Cellular Immunotherapy, by G. Morstyn & W. Sheridan eds, Cambridge University Press, 1996. The choice of cell vehicle and any accompanying elements of the composition is adapted according to the route and device used for administration. The cells may be introduced by injection, catheter, etc. The cells may be frozen at liquid nitrogen temperature and stored for long periods of time and can be used upon thawing. When frozen, the cells will usually be stored in 10% DMSO, 50% FCS, 40% RPMI 1640 medium.

[0240] The pharmaceutical formulations of the invention are optionally packaged in a suitable container with written instructions for the desired purpose. Such formulations may contain a cocktail of retinal differentiation and / or trophic factors in a form suitable for combination with the cells or cell populations of the invention described herein. Such compositions may further include suitable buffers and / or excipients suitable for transfer into an animal.

[0241] The cells or cell populations of the invention described herein can be formulated with a pharma- ceutically acceptable carrier. For example, the cells or cell populations of the invention described herein can be administered alone or as a component of a pharmaceutical preparation. The subject compounds can be formulated for administration in any convenient manner for use in medicine. Pharmaceutical preparations suitable for administration may contain the cells or cell populations of the invention described herein in combination with one or more pharma- ceutically acceptable sterile isotonic aqueous or non-aqueous solutions (e.g., balanced salt solutions (BSS)), dispersions, suspensions or emulsions, or sterile powders, which may be reconstituted into sterile injectable solutions or dispersions immediately before use, which may contain antioxidants, buffers, bacteriostatic agents, solutes or suspending agents or thickening agents. An exemplary pharmaceutical formulation comprises the cells or cell populations of the invention described herein in combination with ALCON® BSS PLUS® (a balanced salt solution containing, per mL, 7.14 mg sodium chloride, 0.38 mg potassium chloride, 0.154 mg calcium chloride dihydrate, 0.2 mg magnesium chloride hexahydrate, 0.42 mg sodium phosphate dibasic, 2.1 mg sodium bicarbonate, 0.92 mg dextrose, 0.184 mg glutathione disulfide (oxidized glutathione), hydrochloric acid and / or sodium hydroxide (to adjust the pH to approximately 7.4) in water).

[0242] When administered, pharmaceutical formulations for use in the present disclosure can be in a pyrogen-free, physiologically acceptable form.

[0243] The formulations comprising the cells or cell populations of the present invention described herein used in the methods described herein can be implanted in a suspension, gel, colloid, slurry, or mixture. Furthermore, the formulations can be encapsulated or injected in vitreous humor in a viscous form for delivery to the site of retinal or choroidal damage. Also, at the time of injection, the cryopreserved cells or cell populations of the present invention described herein can be resuspended with a commercially available balanced salt solution to achieve the desired osmolality and concentration for administration by subretinal injection. The formulations can be administered to areas of the central perimacula that have not been completely lost to disease, which can promote the connection and / or survival of the administered cells.

[0244] The cells or cell populations of the invention described herein can be frozen (cryopreserved) as described herein. Upon thawing, the viability of such cells can be at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% at least 95% or 100% (e.g., at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% at least 95% or about 100% of the cells harvested after glycolysis are viable, or at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% at least 95% or about 100% of the number of cells originally frozen are harvested viable after glycolysis). In some cases, the viability of cells before and after thawing is about 80%. In some cases, at least 90%, or at least 95%, or about 95% of the frozen cells are recovered. The cells may be frozen as single cells or as aggregates.

[0245] The cells or cell populations of the invention described herein can be delivered in a pharma- ceutically acceptable ophthalmic formulation by intraocular injection. For example, when administering the formulation by intravitreal injection, the solution can be concentrated so that a minimized volume can be delivered. The concentration of the injection can be any amount that is effective and non-toxic, depending on the factors described herein. Pharmaceutical formulations of the cells or cell populations of the invention described herein for the treatment of patients can be formulated at a dose of at least about 104 cells / mL. Formulations of the cells or cell populations of the invention described herein for the treatment of patients can be formulated at a dose of at least about 10 3 , 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , or 10 10 It is formulated at a dose of 100 cells / mL.

[0246] Pharmaceutical preparations of the cells of the invention described herein may contain at least about 1,000, 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, or 9,000 cone or cone-like photoreceptor cells. Pharmaceutical preparations of cone or cone-like photoreceptor cells may contain at least about 1×10 4 , 2×10 4 , 3×10 4 , 4×10 4 , 5×10 4 , 6×10 4 , 7×10 4 , 8×10 4 , 9×10 4 , 1×10 5 , 2×10 5 , 3×10 5 , 4×10 5 , 5×10 5 , 6×10 5 , 7×10 5 , 8×10 5 , 9×10 5 , 1×10 6 , 2×10 6 , 3×10 6 , 4×10 6 , 5×10 6, 6×10 6 , 7×10 6 , 8×10 6 , 9×10 6 , 1×10 7 , 2×10 7 , 3×10 7 , 4×10 7 , 5×10 7 , 6×10 7 , 7×10 7 , 8×10 7 , 9×10 7 , 1×10 8 , 2×10 8 , 3×10 8 , 4×10 8 , 5×10 8 , 6×10 8 , 7×10 8 , 8×10 8 , 9×10 8 , 1×10 9 , 2×10 9 , 3×10 9 , 4×10 9 , 5×10 9 , 6×10 9 , 7×10 9 , 8×10 9 , 9×10 9 , 1×10 10 , 2×10 10 , 3×10 10 , 4×10 10 , 5×10 10 , 6×10 10 , 7×10 10 , 8×10 10 , or 9 x 10 10 The pharmaceutical preparation of cone or cone-like photoreceptor cells may comprise at least about 1×10 to 1×10 3 , 1×10 2 ~1×10 4 , 1×10 4 ~1×10 5 , or 1 × 10 3 ~1×10 6The pharmaceutical preparation of cone or cone-like photoreceptor cells may comprise at least about 10,000, 20,000, 25,000, 50,000, 75,000, 100,000, 125,000, 150,000, 175,000, 180,000, 185,000, 190,000, or 200,000 cone or cone-like photoreceptor cells. For example, the pharmaceutical preparation of cone or cone-like photoreceptor cells may comprise at least about 20,000-200,000 cone or cone-like photoreceptor cells in a volume of at least about 50-200 μL. Further, the pharmaceutical preparation of cone or cone-like photoreceptor cells may contain about 50,000 photoreceptors in a volume of 150 μL, about 200,000 cone or cone-like photoreceptor cells in a volume of 150 μL, or at least about 180,000 photoreceptor cells in a volume of at least about 150 μL.

[0247] In the aforementioned pharmaceutical formulations and compositions, the number of cone or cone-like photoreceptor cells, or the concentration of photoreceptor cells, can be determined by counting viable cells and excluding non-viable cells. For example, non-viable photoreceptors can be detected by failure to exclude vital dyes (such as trypan blue) or using functional assays (ability to adhere to culture substrates, phagocytosis, etc.). In addition, the number of photoreceptor cells or the concentration of photoreceptor cells can be determined by counting cells expressing one or more photoreceptor cell markers and / or excluding cells expressing one or more markers indicative of cell types other than photoreceptors.

[0248] Cone or cone-like photoreceptor cells may be formulated for delivery in a pharma- ceutically acceptable ophthalmic vehicle such that the formulation is maintained in contact with the ocular surface for a period of time sufficient to allow the cells to penetrate diseased areas of the eye, such as, for example, the anterior chamber, posterior chamber, vitreous body, aqueous humor, vitreous body, cornea, iris / ciliary body, lens, choroid, retina (e.g., subretinal), sclera, suprachorionic space, conjunctiva, subconjunctival space, extrascleral space, intracorneal space, epicorneal space, synovium, surgically-induced avascular area, or macula.

[0249] The methods described herein may further include monitoring the effectiveness of the treatment or prevention by measuring the subject's electroretinogram response, optic nerve motor acuity threshold, or luminance threshold. The methods may also include monitoring the effectiveness of the treatment or prevention by monitoring the immunogenicity of the cells or the migration of the cells within the eye.

[0250] It should be noted that human cells can be used in human patients as well as animal models or animal patients. For example, human cells can be tested in mouse, rat, cat, dog, or non-human primate models of retinal degeneration. In addition, human cells can be used therapeutically to treat animals in need thereof, such as veterinary medicine. Examples of veterinary subjects or patients include, but are not limited to, dogs, cats, and other companion animals, as well as economically valuable animals such as livestock and horses.

[0251] In addition to the use of in vitro or in vivo generated cone or cone-like photoreceptor cells as described above, the use of gene therapy approaches to reprogram cells in situ or in vivo into cone or cone-like photoreceptor cells is also contemplated.

[0252] As mentioned above, the subject nucleic acids and gene delivery vectors described herein, collectively referred to herein as "the subject compositions", are used for expressing a transgene in a cone cell of an animal. For example, the subject compositions can be used in research, for example, to determine the effect of a gene on the viability and / or function of a cone cell. As another example, the subject compositions may be used in medicine, for example, to treat a cone cell disorder. Thus, in some aspects of the invention, a method is provided for expressing a gene in a cone cell, the method comprising contacting a cone cell with a composition of the present disclosure. In some embodiments, the contacting is performed in vitro. In some embodiments, the contacting is performed in vivo, i.e., the subject compositions are administered to a subject.

[0253] When cells are contacted in vitro with the subject nucleic acids or gene delivery vectors described herein, the cells can be of any mammalian species, e.g., rodent (e.g., mouse, rat, gerbil, squirrel), rabbit, cat, dog, goat, sheep, pig, horse, cow, primate, human.

[0254] When cells are contacted in vitro with the subject nucleic acids or gene delivery vectors described herein, the subject can be any mammal, e.g., a rodent (e.g., mouse, rat, gerbil), rabbit, cat, dog, goat, sheep, pig, horse, cow, or primate.

[0255] The disclosed methods and compositions are used to treat any condition that can be addressed, at least in part, by producing functional cone photoreceptor cells. Thus, the disclosed compositions and methods are used to treat individuals in need of cone cell therapy. By people in need of cone cell therapy, we mean individuals who have or are at risk of developing a cone cell disorder. "Cone cell disorder" means any disorder affecting retinal cone cells, including, but not limited to, ocular vision disorders associated with defects in cone cells, such as cone-specific defects, such as macular dystrophies, such as Stargardt macular dystrophy, cone dystrophy, cone-rod dystrophy, spinocerebellar ataxia type 7, and Bardet-Biedl syndrome-1; and color blindness, partial achromatopsia, blue-cone monochromacy, and protanopia. color vision disorders, including normal, deutan, and tritanopia; and central macular vision disorders (in primates) that can be treated by targeting cone cells, such as age-related macular degeneration, macular telangiectasia, retinitis pigmentosa, diabetic retinopathy, retinal vein occlusion, glaucoma, Sorsby fundus dystrophy, adult vitelliform macular dystrophy, Best's disease, rod-cone dystrophy, Leber's congenital amaurosis, and X-linked retinoschisis.

[0256] Stargardt macular dystrophy. Stargardt macular dystrophy, also known as Stargardt disease and fundus flava, is a genetic form of early-onset macular degeneration that usually causes progressive vision loss up to legal blindness. Onset of symptoms usually appears between the ages of 6 and 30 (average about 16-18 years). Mutations in several genes, including ABCA4, CNGB3, ELOVL4, and PROM1, are associated with the disorder. Symptoms typically develop by the age of 20 and include wavy vision, blind spots, blurred vision, impaired color vision, and difficulty adapting to dim lighting. The primary symptom of Stargardt disease is loss of vision in the range of 20 / 50 to 20 / 200. In addition, those with Stargardt disease are sensitive to glare. Cloudy days are alleviated to some extent. Vision is most significantly reduced when the macula is damaged. This can be observed by fundus examination.

[0257] Cone dystrophies. Cone dystrophies (CODs) are inherited eye disorders characterized by the loss of cone cells. The most common symptoms of cone dystrophies are visual acuity loss (age of onset from late teens to the sixth decade), sensitivity to bright light, and poor color vision. Visual acuity usually deteriorates gradually, but may deteriorate rapidly to 20 / 200. It then declines to "counting fingers" visual acuity in more severe cases. Color vision testing using color test plates (HRR series) reveals many errors on both red-green and blue-yellow plates. The dystrophies are considered primary because subjective and objective abnormalities in cone function are seen before any ophthalmoscopic changes are seen. However, the retinal pigment epithelium (RPE) is rapidly involved, causing retinal dystrophy that primarily involves the macula. Fundus examination with an ophthalmoscopic eye is essentially normal early on in cone dystrophies, with clear macular changes usually occurring after vision loss. The most common type of macular lesion seen during ophthalmoscopy consists of a donut-shaped zone of atrophic pigment epithelium with a bull's-eye image and surrounding a central dark area. In another, less frequent form of cone dystrophy, there is rather diffuse atrophy of the posterior pole with patchy pigment mass deposits in the macular region. Rarely, atrophy of the choriocapillaris and larger choroidal vessels is seen in patients at an early stage. Fluorescein angiography (FA) is a useful adjunct in the workup of individuals suspected of having cone dystrophy and may detect early retinal changes that are too subtle to be seen by ophthalmoscopy. Electroretinogram (ERG) remains the best test for making the diagnosis, as the spectrum of fundus changes is wide and the diagnosis difficult to make at an early stage. Abnormal cone function on the ERG is indicated by a reduced single flash and flicker response when the examination is performed in a well-lit room (photopic ERG). Mutations in several genes, including GUCA1A, PDE6C, PDE6H, and RPGR, are associated with the disorder.

[0258] Spinocerebellar ataxia type 7. Spinocerebellar ataxia is a progressive, degenerative, inherited disease characterized by slowly progressive incoordination of gait, often associated with poor coordination of hand, speech, and eye movements. There are multiple types of SCA, and spinocerebellar ataxia type 7 (SCA-7) differs from most other SCAs in that vision problems may occur in addition to poor coordination. SCA-7 is associated with an autosomal dominant mutation in the ATXN7 / SCA7 gene. When the disease appears before age 40, vision problems, rather than poor coordination, are typically the earliest sign of the disease. Early symptoms include difficulty distinguishing colors and reduced central vision. In addition, symptoms of ataxia (incoordination, slow eye movements, and mild changes in sensation or reflexes) may be detectable. Loss of motor control, slurred speech, and difficulty swallowing become more pronounced as the disease progresses.

[0259] Bardet-Biedl syndrome-1. Bardet-Biedl syndrome-1 (BBS-1) is a pleiotropic disorder with variable expressivity and a wide range of clinical variability observed both within and between families. The main clinical features are childhood-onset rod-cone dystrophy with vision loss preceding night blindness, postaxial polydactyly, truncal obesity that appears in infancy and remains problematic through adulthood, specific learning difficulties in some but not all individuals, male genital hypoplasia and complex female genitourinary malformations, and renal dysfunction that is a major cause of morbidity and mortality. Vision loss is one of the main features of Bardet-Biedl syndrome. Night vision problems become evident by mid-childhood, followed by blind spots that develop in the peripheral vision. Over time, these blind spots enlarge and merge, causing tunnel vision. Most people with Bardet-Biedl syndrome also develop blurred central vision (decreased visual acuity) and become legally blind by adolescence or early adulthood. Bardet-Biedl syndrome can be caused by mutations in at least 14 different genes (often referred to as BBS genes) known or suspected to play important roles in eyelash function, with mutations in BBS1 and BBS10 being the most common.

[0260] Color vision deficiency. Color vision deficiency, or rod monochromacy, is a disorder in which a subject experiences a complete lack of color perception, resulting in the subject seeing only black, white, and shades of gray. Other symptoms include reduced visual acuity, photophobia, nystagmus, small central scotoma, and eccentric fixation. The disorder is often first noticed in children around 6 months of age by photophobic behavior and / or nystagmus. Visual acuity and eye movement stability generally improve (but remain close to 20 / 200) during the first 6 to 7 years of life. Mutations in CNGB3, CNGA3, GNAT2, PDE6C, and PDE6HI have been associated with the disorder.

[0261] Imperfect color vision deficiency is similar to color vision deficiency, but with a lower penetrance. In imperfect color vision deficiency, the symptoms are similar to those of color vision deficiency, except in a reduced form. Individuals with imperfect color vision deficiency have reduced visual acuity, with or without nystagmus or photophobia. Additionally, these individuals show only partial impairment of cone cell function, but still possess rod cells.

[0262] Blue-cone monochromatism. Blue-cone (S-cone) monochromatism (BCM) is a rare X-linked congenital resting cone dysfunction syndrome affecting approximately 1 in 100,000 individuals. Males affected with BCM have no functional long-wavelength-sensitive (L) or mid-wavelength-sensitive (M) rods in the retina due to mutations at the L- and M-opsin gene loci. Color discrimination is severely impaired from birth, and visual acuity is derived from the remaining preserved S-cone and rod photoreceptors. BCM typically presents with reduced visual acuity (6 / 24 to 6 / 60), pendular nystagmus, photophobia, and patients often have myopia. Rod-specific and maximal electroretinograms (ERGs) usually show no clear abnormalities, but 30-Hz cone ERGs cannot be detected. Single-flash photopic ERGs, although small and slow, can often be recorded, and the S-cone ERG is well preserved.

[0263] Color vision deficiency. Color vision deficiency (CVD), or color blindness, is the inability or reduced ability to see color or to detect color differences under normal lighting conditions. Individuals suffering from color blindness can be identified using any of several color vision tests, such as color ERG (cERG), confusion color plates (Ishihara plates, Hardy-Rand-Ritter polychromatic plates), Farnsworth-Munsell 100 hue test, Farnsworth's panel D-15, City University test, and Kollner's rules. Examples of color vision deficiencies include protanopia, deutanopia, and tritanopia. Protanopia includes protanopia (insensitivity to red light) and protanopia (reduced sensitivity to red light) and is associated with mutations in the L-opsin gene (OPN1LW). Deuteranopia includes deuteranopia (insensitivity to green light) and deuteranopia (reduced sensitivity to green light) and is associated with mutations in the M-opsin gene (OPN1MW). Deuteranopia includes tritanopia (insensitivity to blue light) and tritanopia (reduced sensitivity to blue light) and is associated with mutations in the S-opsin gene (OPN1SW).

[0264] Age-Related Macular Degeneration. Age-related macular degeneration (AMD) is one of the leading causes of vision loss in people over the age of 50. AMD primarily affects central vision, which is necessary for detailed tasks such as reading, driving, and facial recognition. Vision loss in this condition results from the gradual deterioration of photoreceptors in the macula. Side (peripheral) vision and night vision are generally not affected.

[0265] Researchers have described two major types of age-related macular degeneration, known as the dry, or "non-exudative," and the wet, or "exudative," or "neovascular," form, both of which may be treated by delivery of a transgene in the context of the subject polynucleotide cassettes.

[0266] Dry AMD is characterized by the accumulation of yellow deposits called drusen between the retinal pigment epithelium and the choroid beneath the macula, which can be observed by fundus photography. This results in slowly progressive vision loss. The condition typically affects vision in both eyes, but vision loss often occurs in one eye before the other. Other changes may include pigmentary changes and RPE atrophy. For example, in certain cases, called central geographic atrophy, or "GA," atrophy of the retinal pigment epithelium and subsequent loss of photoreceptors in the central portion of the eye is observed. Dry AMD has been associated with mutations in CD59 and genes of the complement cascade.

[0267] Wet AMD is an advanced state of dry AMD and occurs in about 10% of dry AMD patients. Pathological changes include retinal pigment epithelial cell (RPE) dysfunction, collection of fluid under the RPE, and choroidal neovascularization (CNV) in the macular region. Fluid leakage, RPE or neuroretinal detachment, and bleeding from ruptured blood vessels can occur in severe cases. Symptoms of wet AMD may include visual distortions such as straight lines that appear wavy or crooked, doorways or road signs that appear tilted, or objects that appear smaller or farther away than they actually are, reduced central vision, reduced intensity or brightness of colors, and clearly defined blurred or blind spots in the visual field. It can develop suddenly and worsen rapidly. Diagnosis may include the use of an Amsler grid to test the subject's central vision defects (macular degeneration may make the straight lines in the grid appear faded, broken, or distorted), fluorescein angiography to look for vascular or retinal abnormalities, and optical coherence tomography to detect retinal swelling or vascular leakage. Many cellular factors are involved in the generation of CNV, including vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), pigment epithelium-derived factor (PEDF), hypoxia-inducible factor (HIF), angiopoietin (Ang), and other cytokines, mitogen-activated protein kinase (MAPK), etc.

[0268] Macular telangiectasia. Macular telangiectasia (MacTel) is a form of pathologically dilated blood vessels (telangiectasia) in the parafoveal region of the macula. It results from the development of fluid-filled cysts that impair the nutrition of photoreceptor cells and permanently destroy vision, leading to tissue deterioration and scarring of retinal structures. There are two types of MacTel: type 1 and type 2. Macular telangiectasia type 2 is a bilateral disease, and its prevalence has recently been shown to be as high as 0.1% in people over 40 years of age. Biomicroscopy may show reduced retinal transparency, crystalline deposits, mildly divergent capillaries, blunted venules, retinal pigment plaques, foveal atrophy, and neovascular complexes. Fluorescein angiography shows transient microaneurysms mainly to the fovea in early stages and diffuse hyperfluorescence in later stages. High-resolution optical coherence tomography (OCT) may reveal destruction of the photoreceptor inner segment-outer segment boundary, hyporeflective cavities at the level of the inner or outer retina, and retinal atrophy at later stages. In macular telangiectasia type 1, the disease almost always occurs in one eye, which distinguishes it from type 2. MacTel does not usually cause complete blindness, but does cause loss of central vision necessary for reading and driving vision, typically over a period of 10 to 20 years.

[0269] Retinitis pigmentosa. Retinitis pigmentosa (RP) is a group of inherited disorders characterized by progressive peripheral vision loss and impaired night vision (night blindness) that may lead to central vision loss. The presenting signs and symptoms of RP vary, but typically include night blindness (night blindness, most commonly the earliest manifestation of RP), vision loss (usually peripheral, but in advanced cases central vision loss), and visual impairment (seeing flashes of lights). Because RP is a conglomeration of many inherited disorders, significant variability in physical findings exists. Ophthalmologic examination includes evaluation of visual acuity and pupillary response, as well as anterior segment, retinal, and fundus examination. In some cases, RP is an aspect of a syndrome, such as syndromes that are also associated with hearing loss (Usher syndrome, Waardenburg syndrome, Alport syndrome, Refsum disease); Kearns-Sayre syndrome (external ophthalmoplegia, ptosis, heart block, and pigmentary retinopathy); abetalipoproteinemia (fat malabsorption, fat-soluble vitamin deficiency, spinocerebellar degeneration, and retinitis pigmentosa); mucopolysaccharidoses (e.g., Hurler syndrome, Scheie syndrome, Sanfilippo syndrome); Bardet-Biedl syndrome (polydactyly, truncal obesity, renal dysfunction, short stature, and pigmentary retinopathy); and neuronal ceroid lipofuscinosis (dementia, encephalocele, and pigmentary retinopathy; the childhood form is known as Jansky-Bielschowski disease, the juvenile form is Vogt-Spielmeyer-Batten disease, and the adult form is Kufs syndrome). Retinitis pigmentosa is most commonly associated with mutations in the RHO, RP2, RPGR, RPGRIP1, PDE6A, PDE6B, MERTK, PRPH2, CNGB1, USH2A, ABCA4, and BBS genes.

[0270] Diabetic Retinopathy. Diabetic retinopathy (DR) is damage to the retina caused by complications of diabetes that can ultimately lead to blindness. Without wishing to be bound by theory, it is believed that hyperglycemia-induced death of intramural pericytes and thickening of the basement membrane cause incompetence of the blood vessel wall. These damages alter the formation of the blood-retinal barrier and also make the retinal blood vessels more permeable.

[0271] There are two stages of diabetic retinopathy: non-proliferative diabetic retinopathy (NPDR) and proliferative diabetic retinopathy (PDR). Non-proliferative diabetic retinopathy is the first stage of diabetic retinopathy and is diagnosed by fundus examination and coexisting diabetes. In cases of reduced vision, a fluorescein angiogram may be performed to visualize the blood vessels behind the eye and any retinal ischemia that may be present. All people with diabetes are at risk of developing NPDR and would therefore be candidates for preventative treatment with the subject vector. Proliferative diabetic retinopathy is the second stage of diabetic retinopathy, characterized by retinal neovascularization, vitreous hemorrhage, and blurred vision. In some cases, fibrovascular proliferation causes tractional retinal detachment. In some cases, blood vessels may also grow into the anterior chamber angle, causing neovascular glaucoma. Individuals with NPDR are at increased risk of developing PDR and would therefore be candidates for preventative treatment with the subject vector.

[0272] Diabetic Macular Edema. Diabetic macular edema (DME) is a progressive, vision-limiting complication of diabetic retinopathy that affects nearly 30% of patients who have had diabetes for at least 20 years and is responsible for the majority of vision loss due to DR. It is due to changes in the retinal microvasculature that compromise the blood-retinal barrier, allowing plasma components to leak into the surrounding retina, resulting in retinal edema. Without wishing to be bound by theory, it is believed that hyperglycemia, persistent alterations in cell signaling pathways, and chronic microvascular inflammation with leukocyte-mediated damage cause chronic retinal microvascular damage, which triggers increased intraocular levels of VEGF, which in turn increases the permeability of the vasculature.

[0273] Patients at risk for developing DME include those who have had diabetes for a long time and experience one or more of the following: severe hypertension (high blood pressure), fluid retention, hypoalbuminemia, or hyperlipidemia. Common symptoms of DME are blurred vision, floaters, double vision, and ultimately blindness if the condition progresses untreated. DME is diagnosed by fundus examination as retinal thickening within two papillary diameters in the center of the macula. Other methods that may be used include optical coherence tomography (OCT) to detect retinal swelling, cystoid edema, and serous retinal detachment; fluorescein angiography, which differentiates and localizes areas of focal and diffuse leakage, thereby guiding the placement of laser photocoagulation if laser photocoagulation is used to treat edema; and color stereo fundus photography, which can be used to assess long-term changes in the retina. Visual acuity may also be measured, particularly to track the progression of macular edema and monitor its treatment following administration of the subject pharmaceutical compositions.

[0274] Retinal Vein Occlusion. Retinal vein occlusion (RVO) is a blockage of the part of the circulation that drains the retina of blood. The blockage can cause backpressure in the capillaries, which can lead to bleeding and leakage of fluid and other components of blood.

[0275] Glaucoma. Glaucoma is a term describing a group of ocular (eye) disorders that cause optic nerve damage and are often associated with increased fluid pressure (intraocular pressure) (IOP) inside the eye. The disorders can be roughly divided into two main categories: "open-angle" and "closed-angle" (or "angle closure") glaucoma. Open-angle glaucoma accounts for 90% of glaucoma cases in the United States. It is painless and has no acute attacks. The only signs are gradually progressive visual field loss and changes in the optic nerve (increased cup-to-disc ratio on funduscopy). Angle-closure glaucoma accounts for less than 10% of glaucoma cases in the United States, but accounts for as many as half of glaucoma cases in other countries, especially in Asia. Approximately 10% of patients with angle-closure present with an acute angle-closure crisis characterized by sudden eye pain, halos around lights, red eyes, very high intraocular pressure (greater than 30 mmHg), nausea and vomiting, sudden loss of vision, and fixed intermediate dilated pupils. In some cases, oval pupils are also associated. Modulating the activity of proteins encoded by DLK, NMDA, INOS, CASP-3, Bcl-2, or Bcl-xl may treat the condition.

[0276] Sorsby Fundus Dystrophy. Sorsby Fundus Dystrophy is an autosomal dominant retinal disease associated with mutations in the TIMP3 gene. Clinically, it presents with early intermediate to peripheral drusen and impaired color vision. Some patients complain of night blindness. Most commonly, the presenting symptom is sudden vision loss appearing in the second to third decade due to untreatable submacular neovascularization. Histologically, there is an accumulation of confluent lipid-containing material 30 pm thick at the level of Bruch's membrane.

[0277] Vitelliform Macular Dystrophy. Vitelliform macular dystrophy is an inherited eye disorder that can cause progressive vision loss. Vitelliform macular dystrophy is associated with the buildup of a fatty yellow pigment (lipofuscin) in cells underlying the macula. Over time, the abnormal buildup of this material can damage cells that are important for clear central vision. As a result, people with this disorder often lose central vision and may have blurred or distorted vision. Vitelliform macular dystrophy does not usually affect side (peripheral) vision or night vision.

[0278] Researchers have described two forms of vitelliform macular dystrophy with similar characteristics. The early-onset form (known as Best's disease) usually appears in childhood. The onset of symptoms and the severity of vision loss vary widely. It is associated with mutations in the VMD2 / BEST1 gene. The adult-onset form (adult vitelliform macular dystrophy) tends to begin later, usually in mid-adulthood, causing vision loss that worsens slowly over time. It is associated with mutations in the PRPH2 gene. Each of the two forms of vitelliform macular dystrophy has characteristic changes in the macula that can be detected during an eye exam.

[0279] Leber's Congenital Amaurosis. Leber's congenital amaurosis (LCA) is a severe retinal dystrophy that typically becomes evident in the first year of life. Visual function is usually poor, often with nystagmus, delayed or nearly absent pupillary response, photophobia, high hyperopia, and keratoconus. Visual acuity is rarely better than 20 / 400. A characteristic finding is the Franschette finger eye phenomenon, consisting of poking, pushing, and rubbing of the eye. The fundus appearance is highly variable. The retina may appear normal initially, but pigmentary retinopathy reminiscent of retinitis pigmentosa is frequently observed later in childhood. The electroretinogram (ERG) is characteristically "undetectable" or severely subnormal. Mutations in the following 17 genes are known to cause LCA: GUCY2D (locus name: LCA1), RPE65 (LCA2), SPATA7 (LCA3), AIPL1 (LCA4), LCAS (LCAS), RPGRIP1 (LCA6), CRX (LCAT), CRB1 (LCA8), NMNAT1 (LCA9), CEP290 (LCA10), IMPDH1 (LCA11), RD3 (LCA12), RDH12 (LCA13), LRAT (LCA14), TULP1 (LCA15), KCNJ13 (LCA16), and IQCB1. Collectively, mutations in these genes are estimated to account for more than half of all LCA diagnoses. At least one other disease locus for LCA has been reported, but the gene is unknown.

[0280] X-linked retinoschisis. X-linked retinoschisis (XLRS) is characterized by symmetric bilateral macular involvement that develops within the first decade of life, in some cases as early as 3 months of age. Fundus examination may reveal areas of macula hiatus (disruptions of the nerve fiber layer of the retina), giving the impression of an axle-like pattern. Peripheral retinal breaks, primarily in the inferior temporal region, occur in approximately 50% of individuals. Affected males typically have visual acuity of 20 / 60 to 20 / 120. Visual acuity often worsens during the first and second decades of life, but then remains relatively stable until the fourth or fifth decade of life. The diagnosis of juvenile X-linked retinoschisis is based on fundus findings, electrophysiological test results, and molecular genetic testing. RS1 is the only gene known to be associated with juvenile X-linked retinoschisis.

[0281] Individuals suffering from or at risk of developing a cone cell disorder can be readily identified using techniques known in the art to detect symptoms of the disorder, including, but not limited to, fundus photography; optical coherence tomography (OCT); adaptive optics (AO); electroretinograms, e.g., ERG, color ERG (cERG); color vision tests, e.g., confusion color plates (Ishihara plates, Hardy-Rand-Ritter polychromatic plates), Farnsworth-Munsell 100 hue test, Farnsworth's panel D-15, City university test, Kollner's rules, etc.; and visual acuity tests, e.g., ETDRS letter test, Snellen visual acuity test, visual field test, contrast sensitivity test, as known by those skilled in the art. Additionally or alternatively, individuals suffering from or at risk of developing a cone cell disorder can be readily identified using techniques for detecting genetic mutations associated with cone cell disorders known in the art, including, but not limited to, PCR, DNA sequence analysis, restriction digestion, Southern blot hybridization, mass spectrometry, etc. In some embodiments, the method includes identifying an individual in need of cone cell therapy. In such cases, any convenient method for determining whether an individual has symptoms of a cone cell disorder or is at risk of developing a cone cell disorder can be utilized to identify an individual in need of cone cell therapy, such as by detecting symptoms described herein or known in the art, by detecting mutations in genes described herein or known in the art, etc.

[0282] Administration When performing an in vivo method, a composition for in vivo reprogramming is typically delivered to the subject's retina in an amount effective to cause expression of the transgene in, for example, retinal glial cells. In some embodiments, the method includes detecting expression of the transgene in cells of the retina, for example, retinal glial cells.

[0283] In a preferred embodiment, the nucleic acid, vector, AAV, medicament according to the present invention can be administered to a subject by direct injection into the bloodstream, nerve, or site requiring treatment, i.e., the eye. For example, the medicament can be injected at least adjacent to the retina. The injection can be intravenous (bolus or injection), or subcutaneous (bolus or injection), or intradermal (bolus or injection), or intravitreal (bolus or injection), or subretinal (bolus or injection).

[0284] Preferably, the nucleic acid, vector of the invention described herein, preferably the AAV vector of the invention described herein, the recombinant AAV of the invention described herein, or the in vitro or ex vivo reprogrammed cell (or a composition containing the cell) is administered directly to the subject's eye (e.g., retina), preferably by injection, more preferably by retinal injection (e.g., subretinal injection), and most preferably by intravitreal injection.

[0285] The composition may be administered to the retina by any suitable method.For example, the composition may be administered intraocularly via intravitreal or subretinal injection.The general method for delivering nucleic acid or vector via intravitreal or subretinal injection may be illustrated by the following brief outline.These examples are merely intended to illustrate certain features of the method, and are not intended to be limiting in any way.

[0286] For subretinal administration, the nucleic acid or vector may be delivered in the form of a suspension that is injected subretinally under direct observation using a surgical microscope. Typically, a volume of 1-200 μL, e.g., 50 μL, 100 μL, 150 μL, or 200 μL, but usually 200 μL or less, of the subject composition is administered by such methods. The procedure may involve vitrectomy followed by injection of the vector suspension into the subretinal space using a fine cannula through one or more small retinal incisions. Briefly, the injection cannula may be sutured in place and normal bulbar volume may be maintained by injection (e.g., of saline) during surgery. Vitrectomy is performed using a cannula of appropriate bore size (e.g., 20-27 gauge), and the volume of vitreous gel that is removed is replaced by injection of saline or other isotonic solution from the injection cannula. Vitrectomy is advantageously performed because (1) its cortical (posterior hyaloid membrane) removal facilitates penetration of the retina with the cannula, (2) its removal and replacement with fluid (e.g., saline) creates space to accommodate intraocular injection of nucleic acid or vector, and (3) its controlled removal reduces the possibility of retinal tears and unplanned retinal detachment.

[0287] For intravitreal administration, the nucleic acid or vector can be delivered in the form of a suspension. First, a local anesthetic is applied to the surface of the eye, followed by a topical antiseptic solution. The eye is held open with or without an instrument, and the nucleic acid or vector is injected through the sclera into the vitreous cavity of the subject's eye under direct observation using a short, narrow, e.g., 30-gauge needle. Typically, a volume of 1-100 μL, e.g., 25 μL, 50 μL, or 100 μL, and usually no more than 100 μL, of the subject compositions may be delivered to the eye by intravitreal injection without removing the vitreous. Alternatively, a vitrectomy may be performed, and the entire volume of the vitreous gel is replaced by injection of the subject compositions. In such cases, up to about 4 mL of the subject compositions may be delivered to, for example, a human eye. Intravitreal administration is generally well tolerated. At the end of the procedure, there may be mild redness at the injection site. There is occasional tenderness, but most patients report no pain. After the procedure, no eye patch or eye shield is needed and activity is not restricted. Antibiotic eye drops may be prescribed for a few days to help prevent infection.

[0288] In practicing the method, the composition is typically delivered to the subject's retina in an amount effective to effect expression of the transgene in cone cells, hi some embodiments, the method includes detecting expression of the transgene in cells of the retina.

[0289] There are many methods for detecting the expression of a transgene, any of which may be used in the subject embodiments. For example, expression may be detected directly, i.e., by measuring the amount of gene product, e.g., at the RNA level, e.g., by RT-PCR, Northern blot, RNAse protection, or at the protein level, e.g., by Western blot, ELISA, immunohistochemistry, etc. As another example, expression may be detected indirectly, i.e., by detecting the effect of the gene product on the viability or function of cone photoreceptors in a subject. For example, if the gene product encoded by the transgene improves the viability of cone cells, expression of the transgene may be detected by detecting the improvement of the viability of cone cells, e.g., by fundus photography, optical coherence tomography (OCT), adaptive optics (AO), etc. If the gene product encoded by the transgene alters the activity of cone cells, expression of the transgene can be detected by detecting changes in the activity of cone cells, for example, by electroretinogram (ERG) and color ERG (cERG); functional adaptive optics; color vision tests, such as confusion color plates (Ishihara plates, Hardy-Rand-Ritter polychromatic plates), Farnsworth-Munsell 100 hue test, Farnsworth's panel D-15, City university test, Kollner's rules, etc.; and visual acuity tests, such as ETDRS letter test, Snellen visual acuity test, visual field test, contrast sensitivity test, etc., as methods for detecting the presence of the delivered polynucleotide. In some cases, both improved viability and altered cone cell function can be detected.

[0290] In some embodiments, the method provides a therapeutic benefit, such as, for example, preventing the onset of a disorder, stopping the progression of a disorder, or reversing the progression of a disorder. In some embodiments, the method includes a step of detecting that a therapeutic benefit has been achieved. Those skilled in the art will understand that such indicators of therapeutic efficacy are applicable to the particular disease to be modified, and will recognize the appropriate detection method to use to measure therapeutic efficacy. For example, therapeutic efficacy in treating retinal degeneration may be observed as a reduction in the rate of retinal degeneration or a halt in the progression of retinal degeneration, and the effect may be observed by comparing the test results after administration of the composition with the test results before administration of the subject composition, for example, by fundus photography, OCT, or AO. As another example, therapeutic efficacy in treating progressive cone dysfunction may be observed as a slowing of the rate of progression of cone dysfunction, a halt to the progression of cone dysfunction, or an improvement in cone function, which effect may be observed, for example, by detecting changes in cone viability and / or function, e.g., by ERG and / or cERG; color vision testing; functional adaptive optics; and / or visual acuity testing, e.g., by comparing test results after administration of a composition with test results before administration of the subject compositions.

[0291] Individual doses are typically doses equal to or greater than that required to produce a measurable effect in a subject, and can be determined based on the pharmacokinetics and pharmacology of the absorption, distribution, metabolism, and excretion ("ADME") of the subject compositions or their by-products, and thus based on the disposition of the compositions in the subject's body. This includes considerations of route of administration and dosage, which can be adjusted for subretinal (applied directly to the site of desired action for a primarily localized effect), intravitreal (applied intravitreally for a pan-retinal effect), or parenteral (applied by a systemic route, e.g., intravenous, intramuscular, etc.) application. Effective amounts of doses and / or dosing schedules can be readily determined empirically from preclinical assays, from safety and escalation and dose ranging studies, and from the relationship of the individual clinician to the patient.

[0292] It will be understood that the invention disclosed and defined herein extends to all alternative combinations of two or more of the individual features mentioned or apparent from the text or drawings, all of these different combinations constituting various alternative aspects of the invention. EXAMPLES

[0293] Here, we have discovered an in vitro method for reprogramming Müller glial cells into cone photoreceptors by transduction with a cocktail of transcription factors called iCone derived (iCone). Furthermore, we demonstrate how to use the iCone factors described herein to perform an in vivo method of reprogramming cells of the eye into cone photoreceptors, which may be used to prevent progressive vision loss associated with diseases that cause photoreceptor degeneration.

[0294] Example 1 - Materials and Methods Generation of reporter cells To establish human MG cell lines with cone reporter, human MG cells MIO-M1 were transduced with OPN1LW / MW DsRed reporter lentivirus at MOI=2 and cells were incubated with the virus overnight in 10% FBS / DMEM medium containing 8μg / mL polybrene. Two days after transduction, cells were selected with 2μg / mL puromycin (ThermoFisher Scientific, A11138-03) for 2 days to generate stable cell lines.

[0295] Genome-wide CRISPRa screening Cone reporter MG cells were used for CRISPRa screening using a pooled lentiviral library of human CRISPR / Cas9 SAM (LentiSAM v2) consisting of 70,290 sgRNAs targeting 23,430 genes. Reporter MG cells were transduced overnight with the pooled lentiviral library in 10% FBS / DMEM medium containing polybrene. After transduction, the virus was removed and fresh 10% FBS / DMEM medium containing TSA (Sigma-Aldrich) was added to the cultures. On day 3 post-transduction, the medium was replaced with NBM / B27 / TSA, which was maintained for the remainder of the reprogramming period up to day 14.

[0296] Flow cytometry sorting was performed on day 14 and DsRed+iCones were isolated using a BD Influx cell sorter (BD Biosciences). DNA extraction was performed on samples and sgRNA was amplified from pooled gDNA of DsRed+ cells. PCR amplification was performed using Q5 High-Fidelity 2X Master Mix (NEB) and monitored using Fast 7500 Real-Time PCR Systems (Applied Biosystems) in the presence of 1X SYBR Green I (Thermo Fisher Scientific). PCR products were then separated on a 2% (w / v) agarose gel and sgRNA was purified with a QIAquick Gel Extraction Kit (QIAGEN). sgRNA sequences were analyzed by Illumina NextSeq 500 (Australian Genome Research Facility). sgRNA distribution was determined using the python script "count_spacers.py" provided by Joung et al., Nat. Protoc., 2017, 12:828-863.

[0297] Gene ontology and network topology analysis Gene ontology was performed using Enrichr (Chen et al., BMC Bioformatics, 2013, 14:128). For network topology analysis, transcription factors were extracted using DAVID v6.8 (LHRI) (Huang et al., Nature Protoc., 2009, 4(1):44-57), and network topology analysis was performed using Cytoscape V3.8 (Shannon et al., Genome Research, 2003, 13:2498-2504).

[0298] Multielectrode array Extracellular field potentials of iPH cells were measured using a microelectrode array (MEA) recording system (Multichannel Systems). MIO-M1 cells were cultured on MEA plates the day before transfection, and recordings were performed 14 days after reprogramming. Data were analyzed using MC Rack software.

[0299] In vitro reprogramming of iCone On day -1, 6 × 10 4Cells were seeded in 12-well plates and cultured in 10% FBS / DMEM. On day 0, cells were co-transduced with a combination of reprogramming transgene lentivirus and cone reporter lentivirus (OPN1LW / MW-DsRed). The next day, fresh medium containing 10 ng / mL TSA (Sigma-Aldrich) was added. On day 3, the culture medium was replaced with NBM / B27 / TSA / T3 containing Neurobasal medium (NBM), B27 (Thermo Fisher Scientific), 10 ng / mL TSA and T3 and maintained until day 14. On day 14, DsRed+ cells were manually quantified to evaluate reprogramming efficiency. RNA was extracted and sent to RNAseq (Australian Genome Research Facility) for iCone characterization. Briefly, the quality of the RNA samples was checked using a bioanalyzer and transcriptome libraries were prepared using the TruSeq Stranded mRNA kit (Illumina). Samples were then processed for 100bp single-end sequencing using an Illumina Novaseq 6000. Transcript level quantification was performed using Salmon, using the human reference transcriptome GRCh38 as an index, to obtain gene-level counts. Gene-level counts were imported using the tximport package and summed using the lengthScaledTPM function, and data are expressed as transcripts per million (TPM) for gene expression analysis.

[0300] In vivo delivery of iCone genes Intravitreal injections of AAV containing iCone factors were performed in P23H3 rats (LaVail et al., Exp. Eye Res., 2018, 167:56-90), which are characterized by progressive photoreceptor degeneration. Seven-week-old P23H-3 rats were injected with AAV (ShH10Y serotype) carrying the iCone gene via intravitreal delivery. Briefly, animals were anesthetized with ketamine (Ilium Ketamil, 20 mg / kg subcutaneously or intramuscularly) and xylazine (Ilium Xylazil-20, 2 mg / kg subcutaneously). Mydriasis was induced by application of 1% tropicamide. Animal body temperature was maintained at 37°C using a heat pad. Intravitreal injections were performed to deliver 3 μl of AAV into the vitreous cavity of the treated eye. Untreated eyes were used as naive controls.

[0301] Electroretinogram (ERG) analysis Dark-adapted full-field retinal photography (ffERG) was performed to assess retinal function before treatment (baseline) and 4 weeks after treatment. ffERG was recorded after placing the animals in the dark for 12 h. Animals were anesthetized with ketamine (20 mg / kg, subcutaneous or intramuscular) and xylazine (2 mg / kg, subcutaneous), after which ketamine was maintained at one-third the original dose as needed. Topical application of sterile saline (0.9%) was used to keep the cornea hydrated during evaluation. Pupils were dilated with 1% tropicamide and 2.5% phenylephrine, and ocular lubricant (HPMC PAA gel) was applied to prevent corneal drying. ffERG was performed using an Espion E2. Retinal responses (average of three measurements) were recorded for stimulus intensities ranging from 0.1 to 30 cd.sm-2. For analysis, post-treatment a-wave and b-wave readings were normalized to baseline (pre-treatment) for each individual eye to assess changes in retinal function after treatment.

[0302] Immunohistochemistry for retina After 4 weeks of treatment, P23H3 rats were euthanized and the posterior eye cups were surgically extracted and fixed in 4% PFA for 2 hours at room temperature. Samples were placed in 10% sucrose for 1 hour, then 20% sucrose for 1 hour, and 30% sucrose overnight at 4° C. The eye cups were placed in a 1 / 1 mixture of 30% sucrose / OCT for 1 hour the following day, then embedded in OCT compound and cryosectioned.

[0303] Standard immunostaining procedures were performed as previously described (Wong et al., Stem Cells, 29(10):1517-27). Briefly, samples were fixed in 4% paraformaldehyde, then blocked with 10% goat serum (Sigma) and permeabilized with 0.1% Triton X-100 (Sigma). Samples were then immunostained with antibodies against recoverin (Millipore), followed by appropriate Alexa Fluor 488 or 568 secondary antibodies (Abcam) and nuclear counterstaining with DAPI (Sigma, 1ug / ml). Samples were imaged using a Zeiss Axio Vert.A1 fluorescent microscope or a Nikon Eclipse TE2000-U. Specificity of staining is confirmed by the absence of signal in isotype controls.

[0304] Example 2 - Experimental setup for genome-wide CRISPRa screening for genes that promote reprogramming of human Müller glia (MG) cells into cones. Figure 1 shows the experimental setup for genome-wide CRISPR activation (CRISPRa) screening to identify genes that promote cone reprogramming. To facilitate viability monitoring and detection of cell reprogramming, we generated human Müller glia (MG) cells (MIO-M1) carrying a fluorescent reporter for the cone marker L / M opsin (OPN1LW / MW-DsRed) using the promoter region for L / M opsin.

[0305] iCone generation For iCone generation, MIO-M1 were transduced with lentivirus containing iCone factors (MOI=10). Virus was incubated overnight in 10% FBS / DMEM medium containing 8 μg / mL polybrene. After transduction, virus was removed and fresh 10% FBS / DMEM medium containing 10 ng / μL TSA was added to the cells. Three days after transduction, medium was replaced with NBM containing B27 and 10 ng / μL TSA. For the remaining reprogramming, medium was replaced with fresh NBM+B27+10 ng / μL TSA every 2 days until day 14.

[0306] Example 3 - Identification of genes that promote iCone reprogramming Using the reporter cell line described above, we performed a genome-wide CRISPRa screen to identify genes that reprogram MG cells into cone photoreceptors called induced cones (iCones). Reporter MG cells were transduced with pooled lentiviruses carrying the CRISPRa library (a SAM library containing 70,290 sgRNAs targeting 23,430 genes). The sequences of the SAM library and sgRNAs are described in Konermann et al., Nature, 2015, 517(7536):583-8, the contents of which are incorporated by reference in their entirety. After 14 days, DsRed+ iCones were sorted using flow cytometry and sequenced to identify candidate genes that promote iCone reprogramming. The pilot screen identified 196 candidate genes, including 31 transcription factors (Figure 2A). Gene ontology analysis of the candidate genes showed that the top four biological processes were related to phototransduction and visual sensing, supporting their role in iCone reprogramming (Figure 2B). Notably, the top hit NEUROG2 (also called NGN2), a key transcription factor in neurodevelopment, is significantly over-represented compared to other candidate genes. Network topology analysis showed significant transcriptional networks among the candidate genes, including NEUROG2 as a core factor (Figure 3). Considering these candidate genes and the list of transcription factors with known roles in retinal development, we shortlisted the following 12 transcription factors for further reprogramming studies: CRX(C), MEF2C(M), THRB(T), RAX(R), NEUROD1(N), RORA(Roa), OTX2(O), PAX6(P), FOXP1(F), ASCL1(A), NEUROG2(Ng), ONECUT1(On).

[0307] Example 4 - Secondary Screening of Transcription Factor Cocktails for iCone Reprogramming and Photoreceptor Characterization We then shortlisted 12 transcription factors with conserved roles in retina / neural development and performed initial screening for iCone reprogramming. Using the CRISPR activation system described above, we induced the expression of the 12 transcription factors in the "cocktail". We identified several factor cocktails that successfully promoted iCone reprogramming within 2 weeks (Figure 6, Table 3).

[0308] To confirm these findings, we performed another primary screen using a lentiviral transgene system to overexpress a cocktail of transcription factors, and then validated the top hits in a secondary screen (Figure 7). These screens identified a multifactor cocktail that could successfully promote iCone reprogramming (Figure 7B).

[0309] [Table 3-1]

[0310] [Table 3-2]

[0311] We performed a screen with different factor cocktails to promote iCone reprogramming (Figure 4). We showed that all 12 candidate iCone genes could be successfully expressed in MG cells using a lentiviral system (data not shown). Characterization of iCones showed expression of a series of cone marker genes, including OPN1L / MW, GNAT2, GRK7, OPN1SW and RXRG (Figures 5A and 5B). These results provide evidence of the feasibility of using cell reprogramming to convert human MG cells to iCone in vitro.

[0312] Furthermore, multielectrode array analysis demonstrated that iCones had functional electrophysiology (Figure 5C). These results provide the first evidence for the feasibility of directly reprogramming human MG cells to generate cones in vitro.

[0313] Example 5 - Reprogramming retinal cells into cone photoreceptor cells in vivo using iCone factors We tested the ability to use iCone factors to prevent vision loss in a rat photoreceptor degeneration model. P23H-3 is a well-established rat retinitis pigmentosa (RP) model caused by rhodopsin mutations (as described in LaVail et al., Exp Eye Res, 2018;167:56-90). P23H-3 undergoes the gradual photoreceptor loss (cones and rods) characteristic of human autosomal dominant RP, making it an ideal model to evaluate iCone reprogramming (Figure 8A) and allow clinical translation to RP patients.

[0314] We utilized adeno-associated virus (AAV) as a delivery system to target Müller glia (MG) cells in the retina (Figure 8B). To ensure specific targeting of MG cells in vivo, we utilized the MG-specific AAV serotype ShH10Y via intravitreal delivery in conjunction with the MG-specific promoter GFAP to drive the expression of the iCone gene. We virally delivered a set of iCone factors (ACNg: Ascl1, Crx, Ngn2) into P23H rats via intravitreal injection and analyzed the visual function of the rats using electroretinograms (ERG) before and after 4 weeks of treatment.

[0315] Four weeks after treatment, electroretinogram (ERG) analysis showed that AAV delivery of iCone factor ACNg to the retina improved visual response in P23H-3 rats (Figure 9). We also observed a local increase in photoreceptor layer thickness (outer nuclear layer, ONL) in iCone factor-treated P23H3 rats compared to sham controls (Figure 10). Taken together, these results support the use of in vivo reprogramming as a novel therapeutic approach to treat photoreceptor degeneration and rescue vision.

Claims

1. 1. A method for reprogramming a source cell, the method comprising increasing protein expression of one or more transcription factors, or biologically active fragments or variants thereof, in the source cell, wherein the source cell is reprogrammed to exhibit at least one characteristic of a target cell; - the source cells are glial cells, preferably human glial cells; - the target cells are photoreceptor cells, - the method, wherein said transcription factor is one or more selected from CRX, ASCL1, NEUROG2, RORA, OTX2, RAX, NEUROD1, PAX6, THRB, MEF2C, FOXP1 and ONECUT1, preferably CRX.

2. 1. An in vitro method for reprogramming a source cell into a cell exhibiting at least one characteristic of a photoreceptor cell, said method comprising: - providing source cells or a cell population comprising source cells; - transfecting said source cells with one or more nucleic acids to increase the expression of one or more genes encoding one or more transcription factors; - culturing said cell or cell population and optionally monitoring said cell or cell population for at least one characteristic of a photoreceptor cell; - the source cells are glial cells, preferably human glial cells; - an in vitro method, wherein said transcription factor is one or more selected from CRX, ASCL1, NEUROG2, RORA, OTX2, RAX, NEUROD1, PAX6, THRB, MEF2C, FOXP1 and ONECUT1, preferably CRX.

3. 3. The method of Claim 2, wherein the one or more nucleic acids comprise an sgRNA for use in a CRISPR activation system to increase expression of genes encoding the one or more transcription factors.

4. The method further comprises: dividing the source cells into at least two of CRX, ASCL1, NEUROG2, RORA, OTX2, RAX, NEUROD1, PAX6, THRB, MEF2C, FOXP1 and ONECUT1; at least three of CRX, ASCL1, NEUROG2, RORA, OTX2, RAX, NEUROD1, PAX6, THRB, MEF2C, FOXP1 and ONECUT1; CRX, ASCL1, NEUROG2, RORA, OTX2, RAX, NEUROD1, PAX6, THRB, MEF2C, FOXP1 and ONECUT1; 3. The method of claim 1 or 2, comprising transfecting with or to increase expression of nucleic acids encoding at least four of ONECUT1, at least five of CRX, ASCL1, NEUROG2, RORA, OTX2, RAX, NEUROD1, PAX6, THRB, MEF2C, FOXP1 and ONECUT1, or at least six of CRX, ASCL1, NEUROG2, RORA, OTX2, RAX, NEUROD1, PAX6, THRB, MEF2C, FOXP1 and ONECUT1.

5. 3. The method of claim 1 or 2, wherein the transcription factors are one or more selected from CRX, ASCL1, NEUROG2, RORA, OTX2, RAX, NEUROD1, PAX6, THRB, MEF2C, FOXP1, and ONECUT1, and the combination of transcription factors results in photoreceptor cells or photoreceptor-like cells with a fold change in opsin mRNA expression that is 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, or 75 fold or more compared to opsin expression in the source cell type.

6. the source cells are glial cells, and the transcription factor, biologically active fragment or variant thereof is (a) CRX, ASCL1 and NEUROG2; (b) ASCL1, NEUROG2 and OTX2; (c) ASCL1, CRX and ONECUT1; (d) ASCL1, CRX and RORA; (e) NEUROD1, NEUROG2 and PAX6; (f) CRX, OTX2 and RAX; (g) ASCL1, CRX and NEUROD1; (h) ASCL1, CRX and THRB; (i) ASCL1, OTX2 and PAX6, (j) NEUROG2 and PAX6; (k) ASCL1, NEUROG2 and PAX6; (l) ASCL1, CRX, and MEF2C; (m) OTX2, RAX, and PAX6; (n) ASCL1, CRX, MEF2C, NEUROD1, OTX2 and THRB, (o) CRX, NEUROG2, THRB and RAX; (p) ASCL1, CRX and OTX2; (q) CRX, NEUROD1 and THRB; (r) ASCL1, NEUROD1, and OTX2; (s) MEF2C, RAX and THRB; (t) MEF2C, PAX6 and OTX2; (u) MEF2C, OTX2 and THRB; (v) MEF2C, OTX2 and RAX; (w) ASCL1, CRX and FOXP1; (x) MEF2C, PAX6 and THRB; (y) MEF2C, NEUROD1 and PAX6; (z) ASCL1, OTX2 and RAX; (aa) ASCL1, CRX and RAX, (bb) CRX, NEUROD1 and OTX2; (cc) CRX, NEUROG2 and OTX2, (dd) CRX, RORA and THRB; (ee) NEUROD1, OTX2 and RAX, (ff) CRX, RAX and THRB, (gg) MEF2C, OTX2 and RORA; (hh) NEUROG2, PAX6 and RAX; (ii) ASCL1, CRX and PAX6; (jj) FOXP1, NEUROG2, PAX6 and THRB, (kk) CRX, NEUROD1 and RAX, (ll) CRX, NEUROG2 and PAX6, (mm) CRX, NEUROD1, OTX2 and RAX, (nn) NEUROG2, OTX2 and PAX6, (oo) CRX and RAX, (pp) PAX6 and RAX, or The method of claim 1 or 2, wherein (qq) CRX, NEUROG2, OTX2 and RAX.

7. 3. The method according to claim 1 or 2, wherein the glial cells are selected from the group consisting of Müller glial (MG) cells, astrocytes and microglia, preferably Müller glial cells.

8. 3. A cell produced by the method of claim 1 or 2, exhibiting at least one characteristic of a photoreceptor cell, preferably a cone cell.

9. A population of cells, wherein at least 1% of the cells exhibit at least one characteristic of a photoreceptor cell, and wherein the cells are produced by the method of claim 1 or 2.

10. A method for producing a transcription factor encoding one or more of the transcription factors defined in claim 1, or a biologically active fragment or variant thereof, or comprising the steps of: (a) CRX, ASCL1 and NEUROG2; (b) ASCL1, NEUROG2 and OTX2; (c) ASCL1, CRX and ONECUT1; (d) ASCL1, CRX and RORA; (e) NEUROD1, NEUROG2 and PAX6; (f) CRX, OTX2 and RAX; (g) ASCL1, CRX and NEUROD1; (h) ASCL1, CRX and THRB; (i) ASCL1, OTX2 and PAX6, (j) NEUROG2 and PAX6; (k) ASCL1, NEUROG2 and PAX6; (l) ASCL1, CRX, and MEF2C; (m) OTX2, RAX, and PAX6; (n) ASCL1, CRX, MEF2C, NEUROD1, OTX2 and THRB, (o) CRX, NEUROG2, THRB and RAX; (p) ASCL1, CRX and OTX2; (q) CRX, NEUROD1 and THRB; (r) ASCL1, NEUROD1, and OTX2; (s) MEF2C, RAX and THRB; (t) MEF2C, PAX6 and OTX2; (u) MEF2C, OTX2 and THRB; (v) MEF2C, OTX2 and RAX; (w) ASCL1, CRX and FOXP1; (x) MEF2C, PAX6 and THRB; (y) MEF2C, NEUROD1 and PAX6; (z) ASCL1, OTX2 and RAX; (aa) ASCL1, CRX and RAX, (bb) CRX, NEUROD1 and OTX2; (cc) CRX, NEUROG2 and OTX2, (dd) CRX, RORA and THRB; (ee) NEUROD1, OTX2 and RAX, (ff) CRX, RAX and THRB, (gg) MEF2C, OTX2 and RORA; (hh) NEUROG2, PAX6 and RAX; (ii) ASCL1, CRX and PAX6; (jj) FOXP1, NEUROG2, PAX6 and THRB, (kk) CRX, NEUROD1 and RAX, (ll) CRX, NEUROG2 and PAX6, (mm) CRX, NEUROD1, OTX2 and RAX, (nn) NEUROG2, OTX2 and PAX6, (oo) CRX and RAX, (pp) PAX6 and RAX, and (qq) CRX, NEUROG2, OTX2 and RAX A nucleic acid comprising a nucleotide sequence encoding one or more of the set of transcription factors of the present invention, or a biologically active fragment or variant thereof.

11. 7. A CRISPR activation system for increasing expression of one or more of the set of transcription factors defined in claim 6, or biologically active fragments or variants thereof.

12. encoding one or more of the transcription factors defined in claim 1 or a biologically active fragment or variant thereof, or (a) CRX, ASCL1 and NEUROG2; (b) ASCL1, NEUROG2 and OTX2; (c) ASCL1, CRX and ONECUT1; (d) ASCL1, CRX and RORA; (e) NEUROD1, NEUROG2 and PAX6; (f) CRX, OTX2 and RAX; (g) ASCL1, CRX and NEUROD1; (h) ASCL1, CRX and THRB; (i) ASCL1, OTX2 and PAX6, (j) NEUROG2 and PAX6; (k) ASCL1, NEUROG2 and PAX6; (l) ASCL1, CRX, and MEF2C; (m) OTX2, RAX, and PAX6; (n) ASCL1, CRX, MEF2C, NEUROD1, OTX2 and THRB, (o) CRX, NEUROG2, THRB and RAX; (p) ASCL1, CRX and OTX2; (q) CRX, NEUROD1 and THRB; (r) ASCL1, NEUROD1, and OTX2; (s) MEF2C, RAX and THRB; (t) MEF2C, PAX6 and OTX2; (u) MEF2C, OTX2 and THRB; (v) MEF2C, OTX2 and RAX; (w) ASCL1, CRX and FOXP1; (x) MEF2C, PAX6 and THRB; (y) MEF2C, NEUROD1 and PAX6; (z) ASCL1, OTX2 and RAX; (aa) ASCL1, CRX and RAX, (bb) CRX, NEUROD1 and OTX2; (cc) CRX, NEUROG2 and OTX2, (dd) CRX, RORA and THRB; (ee) NEUROD1, OTX2 and RAX, (ff) CRX, RAX and THRB, (gg) MEF2C, OTX2 and RORA; (hh) NEUROG2, PAX6 and RAX; (ii) ASCL1, CRX and PAX6; (jj) FOXP1, NEUROG2, PAX6 and THRB, (kk) CRX, NEUROD1 and RAX, (ll) CRX, NEUROG2 and PAX6, (mm) CRX, NEUROD1, OTX2 and RAX, (nn) NEUROG2, OTX2 and PAX6, (oo) CRX and RAX, (pp) PAX6 and RAX, and (qq) CRX, NEUROG2, OTX2 and RAX an adeno-associated virus (AAV) vector, a retrovirus vector, a baculovirus vector, a lentivirus vector, or an mRNA comprising a nucleotide sequence encoding one or more of the set of transcription factors of

13. A recombinant adeno-associated virus (rAAV), comprising: (i) AAV capsid proteins; and (ii) A nucleotide sequence encoding one or more of the transcription factors defined in claim 1, or a biologically active fragment or variant thereof, or (a) CRX, ASCL1 and NEUROG2; (b) ASCL1, NEUROG2 and OTX2; (c) ASCL1, CRX and ONECUT1; (d) ASCL1, CRX and RORA; (e) NEUROD1, NEUROG2 and PAX6; (f) CRX, OTX2 and RAX; (g) ASCL1, CRX and NEUROD1; (h) ASCL1, CRX and THRB; (i) ASCL1, OTX2 and PAX6, (j) NEUROG2 and PAX6; (k) ASCL1, NEUROG2 and PAX6; (l) ASCL1, CRX, and MEF2C; (m) OTX2, RAX, and PAX6; (n) ASCL1, CRX, MEF2C, NEUROD1, OTX2 and THRB, (o) CRX, NEUROG2, THRB and RAX; (p) ASCL1, CRX and OTX2; (q) CRX, NEUROD1 and THRB; (r) ASCL1, NEUROD1, and OTX2; (s) MEF2C, RAX and THRB; (t) MEF2C, PAX6 and OTX2; (u) MEF2C, OTX2 and THRB; (v) MEF2C, OTX2 and RAX; (w) ASCL1, CRX and FOXP1; (x) MEF2C, PAX6 and THRB; (y) MEF2C, NEUROD1 and PAX6; (z) ASCL1, OTX2 and RAX; (aa) ASCL1, CRX and RAX, (bb) CRX, NEUROD1 and OTX2; (cc) CRX, NEUROG2 and OTX2, (dd) CRX, RORA and THRB; (ee) NEUROD1, OTX2 and RAX, (ff) CRX, RAX and THRB, (gg) MEF2C, OTX2 and RORA; (hh) NEUROG2, PAX6 and RAX; (ii) ASCL1, CRX and PAX6; (jj) FOXP1, NEUROG2, PAX6 and THRB, (kk) CRX, NEUROD1 and RAX, (ll) CRX, NEUROG2 and PAX6, (mm) CRX, NEUROD1, OTX2 and RAX, (nn) NEUROG2, OTX2 and PAX6, (oo) CRX and RAX, (pp) PAX6 and RAX, and (qq) CRX, NEUROG2, OTX2 and RAX and an AAV vector comprising a nucleotide sequence encoding one or more of the set of transcription factors of the above, or a biologically active fragment or variant thereof.

14. encoding one or more of the transcription factors defined in claim 1 or a biologically active fragment or variant thereof, or (a) CRX, ASCL1 and NEUROG2; (b) ASCL1, NEUROG2 and OTX2; (c) ASCL1, CRX and ONECUT1; (d) ASCL1, CRX and RORA; (e) NEUROD1, NEUROG2 and PAX6; (f) CRX, OTX2 and RAX; (g) ASCL1, CRX and NEUROD1; (h) ASCL1, CRX and THRB; (i) ASCL1, OTX2 and PAX6, (j) NEUROG2 and PAX6; (k) ASCL1, NEUROG2 and PAX6; (l) ASCL1, CRX, and MEF2C; (m) OTX2, RAX, and PAX6; (n) ASCL1, CRX, MEF2C, NEUROD1, OTX2 and THRB, (o) CRX, NEUROG2, THRB and RAX; (p) ASCL1, CRX and OTX2; (q) CRX, NEUROD1 and THRB; (r) ASCL1, NEUROD1, and OTX2; (s) MEF2C, RAX and THRB; (t) MEF2C, PAX6 and OTX2; (u) MEF2C, OTX2 and THRB; (v) MEF2C, OTX2 and RAX; (w) ASCL1, CRX and FOXP1; (x) MEF2C, PAX6 and THRB; (y) MEF2C, NEUROD1 and PAX6; (z) ASCL1, OTX2 and RAX; (aa) ASCL1, CRX and RAX, (bb) CRX, NEUROD1 and OTX2; (cc) CRX, NEUROG2 and OTX2, (dd) CRX, RORA and THRB; (ee) NEUROD1, OTX2 and RAX, (ff) CRX, RAX and THRB, (gg) MEF2C, OTX2 and RORA; (hh) NEUROG2, PAX6 and RAX; (ii) ASCL1, CRX and PAX6; (jj) FOXP1, NEUROG2, PAX6 and THRB, (kk) CRX, NEUROD1 and RAX, (ll) CRX, NEUROG2 and PAX6, (mm) CRX, NEUROD1, OTX2 and RAX, (nn) NEUROG2, OTX2 and PAX6, (oo) CRX and RAX, (pp) PAX6 and RAX, and (qq) CRX, NEUROG2, OTX2 and RAX A pharmaceutical composition comprising a nucleic acid comprising a nucleotide sequence encoding one or more of the set of transcription factors of the present invention, or a biologically active fragment or variant thereof, or an AAV vector, retroviral vector, baculoviral vector, lentiviral vector or mRNA comprising said nucleic acid, or a recombinant AAV comprising an AAV capsid protein and said AAV vector, and a pharmaceutically acceptable carrier, diluent or excipient.

15. A pharmaceutical composition for use in treating a condition associated with or caused by the degeneration or loss of photoreceptor cells in an individual in need thereof, the composition encoding one or more of the transcription factors defined in claim 1, or biologically active fragments or variants thereof, or comprising: (a) CRX, ASCL1 and NEUROG2; (b) ASCL1, NEUROG2 and OTX2; (c) ASCL1, CRX and ONECUT1; (d) ASCL1, CRX and RORA; (e) NEUROD1, NEUROG2 and PAX6; (f) CRX, OTX2 and RAX; (g) ASCL1, CRX and NEUROD1; (h) ASCL1, CRX and THRB; (i) ASCL1, OTX2 and PAX6, (j) NEUROG2 and PAX6; (k) ASCL1, NEUROG2 and PAX6; (l) ASCL1, CRX, and MEF2C; (m) OTX2, RAX, and PAX6; (n) ASCL1, CRX, MEF2C, NEUROD1, OTX2 and THRB, (o) CRX, NEUROG2, THRB and RAX; (p) ASCL1, CRX and OTX2; (q) CRX, NEUROD1 and THRB; (r) ASCL1, NEUROD1, and OTX2; (s) MEF2C, RAX and THRB; (t) MEF2C, PAX6 and OTX2; (u) MEF2C, OTX2 and THRB; (v) MEF2C, OTX2 and RAX; (w) ASCL1, CRX and FOXP1; (x) MEF2C, PAX6 and THRB; (y) MEF2C, NEUROD1 and PAX6; (z) ASCL1, OTX2 and RAX; (aa) ASCL1, CRX and RAX, (bb) CRX, NEUROD1 and OTX2; (cc) CRX, NEUROG2 and OTX2, (dd) CRX, RORA and THRB; (ee) NEUROD1, OTX2 and RAX, (ff) CRX, RAX and THRB, (gg) MEF2C, OTX2 and RORA; (hh) NEUROG2, PAX6 and RAX; (ii) ASCL1, CRX and PAX6; (jj) FOXP1, NEUROG2, PAX6 and THRB, (kk) CRX, NEUROD1 and RAX, (ll) CRX, NEUROG2 and PAX6, (mm) CRX, NEUROD1, OTX2 and RAX, (nn) NEUROG2, OTX2 and PAX6, (oo) CRX and RAX, (pp) PAX6 and RAX, and (qq) CRX, NEUROG2, OTX2 and RAX or a biologically active fragment or variant thereof, or an AAV vector, retroviral vector, baculoviral vector, lentiviral vector or mRNA comprising said nucleic acid, or a recombinant AAV comprising an AAV capsid protein and said AAV vector, or a cell comprising said nucleic acid, said AAV vector or said recombinant AAV, and a pharmaceutically acceptable carrier, diluent or excipient; the condition associated with or caused by degeneration or loss of photoreceptor cells is any one of retinitis pigmentosa, age-related macular degeneration, choroideremia, and diabetic retinopathy; or the condition associated with or caused by degeneration or loss of photoreceptor cells is a cone cell disorder, such as color vision impairment, macular dystrophy, or central macular vision impairment; Preferably, the color vision defect is selected from the group consisting of achromatopsia, blue-cone monochromacy, protanopia, deutanopia, and tritanopia; Preferably, the macular dystrophy is selected from the group consisting of Stargardt's macular dystrophy, cone dystrophy, spinocerebellar ataxia type 7, and Bardet-Biedl syndrome-1; Preferably, the central macular vision disorder is selected from the group consisting of age-related macular degeneration, macular telangiectasia, retinitis pigmentosa, diabetic retinopathy, retinal vein occlusion, glaucoma, choroideremia, Sorsby fundus dystrophy, adult vitelliform macular dystrophy, Best's disease, Leber's congenital amaurosis, and X-linked retinoschisis.

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