WO2021253078A1

WO2021253078A1 - Process to produce photoreceptor cells

Info

Publication number: WO2021253078A1
Application number: PCT/AU2021/050614
Authority: WO
Inventors: Ching-Bong WONG; Sandy Shen-Chi HUNG; Alex W HEWITT
Original assignee: University of Melbourne; Centre for Eye Research Australia
Current assignee: University of Melbourne; Centre for Eye Research Australia
Priority date: 2020-06-15
Filing date: 2021-06-15
Publication date: 2021-12-23
Anticipated expiration: 2022-12-15

Abstract

The present invention also relates to methods and compositions for direct reprogramming (i.e. transdifferentiation or cellular reprogramming) of a source cell to a cell having characteristics of a photoreceptor cell.

Description

Process to produce photoreceptor cells

Field of the invention

The invention relates to methods and compositions for converting one cell type to another cell type. Specifically, the invention relates to transdifferentiation of a cell to a photoreceptor cell.

Related application

This application claims priority from Australian provisional application AU 2020901972, the entire contents of which are hereby incorporated by reference.

Background of the invention

Cell-based regenerative therapy requires the generation of specific cell types for replacing tissues damaged by injury, disease or age. Embryonic stem cells (ESC) have the potential to differentiate in every cell type from the (human) body and have therefore been extensively studied as a source for replacement therapy. However, ESC cannot be derived in a patient-specific fashion since they are established from cultured blastocysts. Therefore, immune rejection and ethical concerns are the main barriers that prevent the transfer of the ESC technology, and in particular of human ESC technology, to clinical applications.

Cell-replacement therapies have 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-matched cells would also pose less risk for rejection after transplantation. Moreover, these cells would manifest less tumorigenicity since they are terminally differentiated.

Trans-differentiation, the process of converting from one cell type to another without going through a pluripotent state, may have great promise for regenerative medicine but has yet to be reliably applied. Although it may be possible to switch the phenotype of one somatic cell type to another, the elements required for conversion are difficult to identify and in most instances unknown. The identification of factors to directly reprogram the identity of cell types is currently limited by, amongst other things, the cost of exhaustive experimental testing of plausible sets of factors, an approach that is inefficient and unscalable.

Photoreceptor cells, also known simply as photoreceptors, are light-sensing cells within the retina that form the basis of human vision. The degeneration of photoreceptors is a central hallmark of many blinding diseases, including retinitis pigmentosa, age-related macular degeneration, choroideremia and diabetic retinopathy. These diseases affect millions worldwide and results in a significant socio-economic burden on our healthcare system. Critically, there is no cure to blindness once the photoreceptors in the eye are lost. Also, at the late stages of these retinal degenerative diseases, there are often insufficient remaining photoreceptors that can be targeted for pharmacological treatment. In this regard, regenerative medicine represents a highly attractive approach to address this issue.

There is a need for a new and/or improved method for generating cells and cell populations, particularly photoreceptor cells, for use in research and therapeutic applications,

Reference to any prior art in the specification is not an acknowledgment or suggestion that this prior art forms part of the common general knowledge in any jurisdiction or that this prior art could reasonably be expected to be understood, regarded as relevant, and/or combined with other pieces of prior art by a skilled person in the art.

Summary of the invention

The present invention relates to in vitro or ex vivo methods and compositions for direct reprogramming (i.e. transdifferentiation or cellular reprogramming) of a source cell to a cell having characteristics of a photoreceptor cell.

In one aspect, the present invention provides an in vitro or ex vivo method for reprogramming a source cell, the method comprising increasing the protein expression of one or more transcription factors, or variant thereof, in the source cell, wherein the source cell is reprogrammed to exhibit at least one characteristic of a target cell, wherein:

- the source cell is a glial cell, - the target cell is a photoreceptor cell; and

- the transcription factors are one or more of those selected from ASCL1, NEUROD1, NRL, NR2E3, RAX, RORB, OTX2, CRX and PAX6.

Preferably, the glial cell is selected from the group consisting of a Muller glial (MG) cell, an astrocyte and a microglia.

The photoreceptor cells may be rod photoreceptor cells or cone photoreceptor cells. Preferably, the photoreceptor cells are rod photoreceptor cells.

In another aspect, the present invention provides a method of generating a cell exhibiting at least one characteristic of a photoreceptor cell from a source cell, the method comprising:

- increasing the amount of one or more transcription factors, or variant thereof, in the source cell; and

- culturing the source cell for a sufficient time and under conditions to allow differentiation to a photoreceptor cell; thereby generating the cell exhibiting at least one characteristic of a photoreceptor cell from a source cell, wherein:

- the source cell is a glial cell, and

Preferably, the glial cell is selected from the group consisting a Muller glial (MG) cell, an astrocyte and a microglia.

In another aspect, the present invention provides a method for reprogramming a source cell to a cell that exhibits at least one characteristic of a photoreceptor cell, the method comprising:

- providing a source cell, or a cell population comprising a source cell;

- transfecting said source cell with one or more nucleic acids comprising a nucleotide sequence that encodes one or more transcription factors; and - culturing said cell or cell population, and optionally monitoring the cell or cell population for at least one characteristic of a photoreceptor cell, wherein:

- the source cell is a glial cell, and

Further still, the present invention provides an in vitro method for reprogramming a source cell to a cell that exhibits at least one characteristic of a photoreceptor cell, the method comprising:

- providing a source cell, or a cell population comprising a source cell;

- transfecting said source cell with one or more nucleic acids for increasing the expression of one or more genes encoding one or more transcription factors; and

- culturing said cell or cell population, and optionally monitoring the cell or cell population for at least one characteristic of a photoreceptor cell, wherein:

- the source cell is a glial cell and

Preferably, the one or more nucleic acids comprise sgRNAs for use in a CRISPR activation system for increasing the expression of the genes encoding the transcription factors. The sgRNA may be any sgRNA for increasing expression of one or more of ASCL1, NEUROD1, NRL, NR2E3, RAX, RORB, OTX2, CRX and PAX6. The sgRNA may be one or more of those described herein.

In any aspect of the present invention, the glial cell is selected from the group consisting of a Muller glial (MG) cell, an astrocyte and a microglia.

In any aspect of a method of the invention described herein, the source cell is a Muller glial cell, and the transcription factors, or variants thereof, are:

(a) ASCL1 , NEUROD1 , NRL, NR2E3, RAX, RORB, OTX2, CRX and PAX6; (b) ASCL1 , NEUROD1 and NRL;

(c) ASCL1 , NRL and RAX;

(d) ASCL1 , NRL and CRX;

(e) ASCL1 , NRL, RAX and CRX; (f) ASCL1 , NRL and 0TX2;

(g) ASCL1 , NRL and NR2E3;

(h) ASCL1 , NRL and RORB;

(i) RORB, OTX2, CRX and NRL;

0 ASCL1 , NRL, RORB and NR2E3; (k) ASCL1 , NRL, CRX and OTX2;

(L) ASCL1 , NEUROD1 , NRL and CRX;

(m) ASCL1 , NEUROD1 , NRL and NR2E3;

(n) NR2E3, OTX2, RAX and NEUROD1 ;

(o) OTX2; (p) ASCL1 , NRL, NR2E3 and CRX;

(q) ASCL1 , NEUROD1 , NRL and RORB;

(r) ASCL1 , NEUROD1 , NRL and OTX2;

(s) RAX;

(t) OTX2, RAX and NEUROD1; (u) ASCL1 , NRL, NR2E3 and RAX;

(v) RORB; (w) NR2E3, OTX2, CRX, RAX and NEUROD1 ;

(x) ASCL1 ;

(y) NEUROD1;

(z) PAX6; (aa) NEUROD1, CRX, NR2E3 and RAX;

(bb) CRX;

(cc) ASCL1 , NRL, RORB and OTX2;

(dd) NRL;

(ee) ASCL1 , NEUROD1 , NRL, RAX and NR2E3; (ff) ASCL1 , NEUROD1, NRL, NR2E3 and CRX;

(gg) ASCL1 , NRL, RORB and CRX;

(hh) NR2E3;

(ii) CRX, RAX and NEUROD1; or

(jj) CRX, OTX2 and NRL. In any aspect of a method of the invention described herein, the source cell is a

Muller glial cell, and the transcription factors, or variants thereof, are:

(a) ASCL1 , NEUROD1 , NRL, NR2E3, RAX, RORB, OTX2, CRX and PAX6;

(b) ASCL1 , NEUROD1 and NRL;

(c) ASCL1 , NRL and RAX; (d) ASCL1 , NRL and CRX;

(e) ASCL1 , NRL, RAX and CRX;

(f) ASCL1 , NRL and OTX2; (g) ASCL1 , NRL and NR2E3;

(h) ASCL1 , NRL and RORB;

(i) RORB, OTX2, CRX and NRL;

G) ASCL1 , NRL, RORB and NR2E3; (k) ASCL1 , NRL, CRX and OTX2;

(L) ASCL1 , NEUROD1 , NRL and CRX;

(m) ASCL1 , NEUROD1 , NRL and NR2E3;

(n) NR2E3, OTX2, RAX and NEUROD1 ;

(o) OTX2; (p) ASCL1 , NRL, NR2E3 and CRX;

(q) ASCL1 , NEUROD1 , NRL and RORB;

(r) ASCL1 , NEUROD1 , NRL and OTX2;

(s) RAX;

(t) OTX2, RAX and NEUROD1; (u) ASCL1 , NRL, NR2E3 and RAX;

(v) RORB; or

(w) NR2E3, OTX2, CRX, RAX and NEUROD1.

(a) ASCL1 , NEUROD1 , NRL, NR2E3, RAX, RORB, OTX2, CRX and PAX6;

(b) ASCL1 , NEUROD1 and NRL; (c) ASCL1, NRL and RAX;

(d) ASCL1, NRL and CRX;

(e) ASCL1 , NRL, RAX and CRX;

(f) ASCL1, NRL and 0TX2; (g) ASCL1, NRL and NR2E3;

(h) ASCL1, NRL and RORB;

(i) RORB, OTX2, CRX and NRL;

G) ASCL1 , NRL, RORB and NR2E3;

(k) ASCL1 , NRL, CRX and OTX2; (I) ASCL1, NEUROD1, NRL and CRX; or

(m) ASCL1 , NEUROD1 , NRL and NR2E3.

(a) ASCL1 , NEUROD1 , NRL, NR2E3, RAX, RORB, OTX2, CRX and PAX6; (b) ASCL1, NEUROD1 and NRL; or

(c) ASCL1, NRL and RAX.

Preferably, the at least one characteristic of the photoreceptor cell is up-regulation of any one or more target cell markers and/or change in cell morphology. Relevant markers are described herein and known to those in the art. Exemplary markers for the photoreceptor cells include:

- RHO;

- MY07A;

- PDE6B;

- CNGB1; - NR2E3;

- ROM1;

- MEF2C;

- ELOVL4;

- NRL;

- GNAT1;

- CNGA1;

- SAG;

- GNGT1;

- an electrophysiological response in a photopic condition, for example, as described in the Examples.

Markers RHO, MY07A, PDE6B, CNGB1, NR2E3, ROM1, MEF2C, and ELOVL4 are also markers of rod photoreceptor cells.

Additional examples of photoreceptor markers include the opsins that are light detecting molecules. For example, rhodopsin (rod photoreceptor cells), red / green opsin (cone photoreceptor cells), blue opsin (cone photoreceptor cells), and recoverins (rod photoreceptor cells, cone photoreceptor cells).

In any aspect, the combination of transcription factors one or more of those selected from ASCL1, NEUROD1, NRL, NR2E3, RAX, RORB, OTX2, CRX and PAX6, wherein the combination results in a photoreceptor, or photoreceptor-like, cells with a fold increase in RHO mRNA expression of equal to, or greater than, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70 or 75 fold compared to the RHO expression in the source cell type. Preferably, the fold increase is equal to, or greater than, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70 or 75 fold compared to the RHO expression in the source cell type. Most preferably, the fold increase is equal to, or greater than, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70 or 75 fold compared to the RHO expression in the source cell type.

In any aspect of the present invention, the source cell is a human cell. Where the source cell is a Muller glial cell, it may be a human Muller glial cell.

Typically, conditions suitable for photoreceptor cell differentiation include culturing the cells for a sufficient time and in a suitable medium. A sufficient time of culturing 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 shown in Table 2.

In any aspect of the present invention, the cells may be contacted with Trichostatin A during the transfecting or culturing step.

In another aspect, the present invention also provides a cell exhibiting at least one characteristic of a photoreceptor cell produced by a method as described herein.

In any method described herein, the method may further include the step of expanding the cells exhibiting at least one characteristic of a photoreceptor cell to increase the proportion of cells in the population exhibiting at least one characteristic of a photoreceptor cell. The step of expanding the cells may be in culture for a sufficient time and under conditions for generating a population of cells as described below.

In any method described herein, the method may further include the step of administering the cells, or cell population including a cell, exhibiting at least one characteristic of a photoreceptor cell, to an individual.

The present invention also provides a population of cells, wherein at least 1% of cells exhibit at least one characteristic of a photoreceptor cell and those cells are produced by a method as described herein. Preferably, at least 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 a photoreceptor cell.

The present invention also relates to kits for producing a cell exhibiting at least one characteristic of a photoreceptor cell as disclose herein. In some embodiments, a kit comprises one or more nucleic acids having one or more nucleic acid sequences encoding a transcription factor described herein or variant thereof, including the specific combinations referred to in (a) to (jj) herein. Preferably, the kit can be used with a source cell referred to herein. In some embodiments, the kit further comprises instructions for reprogramming a source cell to a cell exhibiting at least one characteristic of a photoreceptor cell according to the methods as disclosed herein. Preferably, the present invention provides a kit when used in a method of the invention described herein.

In another aspect, the present invention relates to a composition comprising at least one source cell as described herein and at least one agent which increases the expression of genes encoding one or more transcription factors in the source cell. Further, the transcription factor may be any one or more described herein, including the combinations reference to in (a) to (jj) herein.

Typically, the gene expression, or amount, of a transcription factor as described herein is increased by contacting the cell with an agent which increases the expression of the transcription factor. Preferably, the agent is selected from the group consisting of: a nucleotide sequence, a protein, an aptamer and small molecule, ribosome, RNAi agent and peptide-nucleic acid (PNA) and analogues or variants thereof. Preferably, the agent is exogenous. In a preferred embodiment, the agent or agents are CRISPR components, such as those described herein, that induce endogenous gene activation. For example, a CRISPR activation system, and components thereof including sgRNAs, such as that described herein, is contemplated as an agent that increases the expression of one or more transcription factors.

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

The gene expression, or amount, of a transcription factor as described herein may also be increased by introducing at least one nucleic acid (such as an sgRNA) for use in a CRISPR activation system, for increasing the expression of the gene encoding the transcription factor.

Preferably, the nucleic acid further includes a heterologous promoter. Preferably, the nucleic acid is in a vector, such as a viral vector or a non- viral vector. Preferably, the vector is a viral vector comprising a genome that does not integrate into the host cell genome. The viral vector may be a retroviral vector or a lentiviral vector. In another aspect, the present invention relates to a nucleic acid or vector comprising a nucleic acid as described herein that may include nucleotide sequences encoding one or more transcription factors as described herein. Preferably, the nucleic acid or vector encodes one or more sets of transcription factors as described herein, including in (a) through to (jj) above, and Table 4 below. In one embodiment, the nucleic acid or vector comprises one or more of the sequences referred to 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 as described herein.

In another aspect, the present invention relates to a CRISPR activation system for increasing the expression of the gene encoding one or more of transcription factors described herein. Preferably the CRISPR activation system results in increasing the expression of one or more sets of transcription factors as described herein, including in (a) through to (jj) above, and Table 4 below. In one embodiment, the CRISPR activation system comprises the sgRNAs described herein, including Table 3.

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

In any aspect of the present invention, the method as described herein may have one or more, or all, steps performed in vitro or ex vivo.

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

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

In another aspect, the present invention provides a cell or cell population generated in vitro or ex vivo by any method described herein for use in the treatment of a condition associated with or caused by degeneration, or loss, of photoreceptor cells in an individual in need thereof. In any aspect, the condition associated with or cause by degeneration, or loss, of photoreceptor cells is any one of retinitis pigmentosa, age-related macular degeneration, choroideremia and diabetic retinopathy.

As used herein, except where the context requires otherwise, the term "comprise" and variations of the term, such as "comprising", "comprises" and "comprised", are not intended to exclude further additives, components, integers or steps.

Further aspects of the present 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.

Brief description of the drawings

Figure 1. A. Fold change gene expression following CRISPR activation to induce expression of 9 candidate transcription factors in human MG cells (MIOMI) using single sgRNA, or multiplex expression of 9 sgRNA. n = 3, error bars = SEM. B. RHO mRNA expression (fold change relative to mock) is shown for 125 reprogramming experiments to identify transcription factor cocktails that reprogram human MG cells into induced photoreceptor cells (iPH) (Arrows indicate: ANNr = ASCL1+NEUROD1+NRL; ANrR = ASCL1+NRL+RAX; 9 genes = ASCL1+NEUROD1+CRX+OTX2+NRL+RAX+ RORB+NR2E3PAX6). Results represent mean of technical triplicates ± SEM. C. RHO mRNA expression (fold change relative to mock) for selected reprogramming conditions as listed in Table 4.

Figure 2. A. and B. Fluorescence microscopy images of derived iPH produced according to the invention. A. Staining with RHO and DAPI indicates that the iPH are positive for rod marker RHO. B. Staining with rod marker PDE6B also indicates that the derived iPH are positive for this marker. C. Violin plot of multielectrode array of mock control and iPH. Pre-illumination and post-illumination (left- and right-hand sides, respectively, for mock and iPH samples), demonstrate that the iPH display functional electrophysiological responses in photopic conditions.

Figure 3. A. Results of single cell RNAseq indicate the presence of an iPH subpopulation (arrow) in which all 9 rod markers are upregulated. C1 - C8 represent clusters identified by single cell transcriptomics and represent subpopulations within the reprogrammed culture. B. Principal component analysis of transcriptomics is expected to induce a transcriptional shift from MG cells to rods. C. Using a single cell human retinal gene atlas as a benchmark, the pilot scRNAseq data captured various reprogramming stages of iPH from MG cells to rod photoreceptors specifically (marked line).

Detailed description of the embodiments

It will be understood that the invention disclosed and defined in this specification 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.

Reference will now be made in detail to certain 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.

One skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which could be used in the practice of 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 in this specification 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.

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

Currently there is no appropriate human photoreceptor cell line available, which represent a bottleneck in the study of retinal diseases. Thus, it is critical to develop good in vitro models for human photoreceptors for the retinal research field, as some questions regarding the unique nature of the human visual system cannot be answered by animal models. This research described herein is the development of direct reprogramming method to generate human photoreceptors in vitro. Critically, the direct reprogramming method is much faster (~2 weeks) compared to iPSC generation and differentiation (~3-6 months), making it more cost-effective for generating human photoreceptors in vitro. The derived human photoreceptors will provide both a better in vitro model to study retinal biology and diseases, providing a platform for drug testing in a clinically relevant cell type, as well as a cellular source for tissue engineering and transplantation for cell therapy.

The present invention provides compositions and methods for direct reprogramming or transdifferentiation of source cells to target cells, without the source cell becoming an induced pluripotent stem cell (iPS) intermediately prior to becoming a target cell. In comparison to iPS cell technology, transdifferentiation is highly efficient and poses a very low risk of teratoma formation for downstream applications.

The process of reprogramming a cell alters the type of progeny a cell can produce and includes transdifferentiation. Transdifferentiation of one somatic cell provides a cell exhibiting at least one characteristic of another somatic cell type.

A source cell may be any cell type described herein, including a somatic cell or a diseased somatic cell. The somatic cell may be an adult cell or a cell derived from an adult. The diseased cell may be a cell displaying one or more detectable characteristics of a disease or condition, for example the diseased cell may be a cancer cell displaying one or more clinical or biochemical markers of a cancer. Examples of source cells include glial cells, such as a Muller glial (MG) cell, an astrocyte and a microglial cell.

As used herein, the term "somatic cell" refers to any cell forming the body of an organism, as opposed to germline cells. In mammals, germline cells (also known as "gametes") are the spermatozoa and ova which fuse during fertilization to produce a cell called a zygote, from which the entire mammalian embryo develops. Every other cell type in the mammalian body — apart from the sperm and ova, the cells from which they are made (gametocytes) and undifferentiated stem cells — is a somatic cell: internal organs, skin, bones, blood, and connective tissue are all made up of somatic cells. In some embodiments the somatic cell is a "non-embryonic somatic cell", by which is meant a somatic cell that is not present in or obtained from an embryo and does not result from proliferation of such a cell in vitro. In some embodiments the somatic cell is an "adult somatic cell", by which is meant a cell that is present in or obtained from an organism other than an embryo or a fetus or results from proliferation of such a cell in vitro. The somatic cells may be immortalized to provide an unlimited supply of cells, for example, by increasing the level of telomerase reverse transcriptase (TERT). For example, the level of TERT can be increased by increasing the transcription of TERT from the endogenous gene, or by introducing a transgene through any gene delivery method or system.

Unless otherwise indicated the methods for reprogramming somatic cells can be performed in vitro, where in vitro is practiced using isolated somatic cells maintained in culture.

Suitable somatic cells are receptive, or can be made receptive using methods generally known in the scientific literature, to uptake of transcription factors including genetic material encoding the transcription factors. Uptake-enhancing methods can vary depending on the cell type and expression system. Exemplary conditions used to prepare receptive somatic cells having suitable transduction efficiency are well-known by those of ordinary skill in the art.

The term "isolated cell" as used herein refers to a cell that has been removed from an organism in which it was originally found or a descendant 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 re-introduced into the organism from which it (or the cell from which it is descended) was isolated.

The term "isolated population" with respect to an isolated population of cells as used herein, 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 were isolated or enriched from.

The term "substantially pure", with respect to a particular cell population, refers 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, with respect to the cells making up a total cell population. Recast, the terms "substantially pure" or "essentially purified", with regard to a population of target cells, refers to a population of cells that contain fewer than about 20%, more preferably fewer than about 15%, 10%, 8%, 7%, most preferably fewer than about 5%, 4%, 3%, 2%, 1%, or less than 1%, of cells that are not target cells or their progeny as defined by the terms herein.

A source cell is determined to be converted to a target cell, or become a target- like cell, by a method of the invention when it displays at least one characteristic of the target cell type, i.e. a photoreceptor cell. For example, a human Muller glial will be identified as converted to a photoreceptor-like cell, when a cell displays at least one characteristic of the photoreceptor cell type. Typically, a cell will display 1, 2, 3, 4, 5, 6, 7, 8 or more characteristics (or markers) of a photoreceptor cell. For example, where the target cell is a photoreceptor cell, a cell is identified or determined to be a photoreceptor-like cell when up-regulation of any one or more photoreceptor cell markers and/or change in cell morphology is detectable, preferably, the increase in RHO mRNA expression. Other examples of photoreceptor markers include RHO, MY07A, PDE6B, CNGB1, NR2E3, ROM1, MEF2C, ELOVL4, NRL, GNAT1, CNGA1, SAG, GNGT1, an electrophysiological response in a photopic condition, for example, as described in the Examples. Markers RHO, MY07A, PDE6B, CNGB1, NR2E3, ROM1, MEF2C, and ELOVL4 are also markers of rod photoreceptor cells. Additional examples of photoreceptor markers include the opsins that are light-detecting molecules. For example, rhodopsin (rod photoreceptor cells), red / green opsin (cone photoreceptor cells), blue opsin (cone photoreceptor cells), and recoverins (rod photoreceptor cells, cone photoreceptor cells). In any aspect of the invention, the target cell characteristic may be determined by analysis of cell morphology, gene expression profiles, activity assay, protein expression profile, surface marker profile, or differentiation ability. Examples of characteristics or markers include those that are described herein and those known to the skilled person.

The transcription factors referred to herein are referred to 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. Pp. D749-D755) version 83. Also contemplated for use in the invention is any homolog, ortholog or paralog of a transcription factor referred to herein.

The skilled person will appreciate that this information may be used in performing the methods of the present invention, for example, for the purposes of providing increased amounts of transcription factors in source cells, or providing nucleic acids or the like for recombinantly expressing a transcription factor in a source cell.

Table 1 : Accession numbers identifying nucleotide sequences and amino acid sequences of transcription factors and proteins referred to herein.

The term a "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. The variant could be a fragment of full length polypeptide or a naturally occurring splice variant. The variant could be a polypeptide at least 70%, 80%, 85%, 90%, 95%, 98%, or 99% identical to a fragment of the polypeptide, wherein the fragment is at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 98%, or 99% as long as the full length wild type polypeptide or a domain thereof has a functional activity of interest 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 in the sequence and extending toward the C-terminus. Variations known in the art to eliminate or substantially reduce the activity of the protein are preferably avoided. In some embodiments, the variant lacks an N- and/or C-terminal portion of the full length polypeptide, e.g., up to 10, 20, or 50 amino acids from either terminus is lacking. In some embodiments the polypeptide has the sequence of a mature (full length) polypeptide, by which is meant a polypeptide that has had one or more portions such as a signal peptide removed during normal intracellular proteolytic processing (e.g., during co-translational or post-translational processing). In some embodiments wherein the protein is produced other than by purifying it from cells that naturally express it, the protein is a chimeric polypeptide, by which is meant that it contains portions from two or more different species. In some embodiments wherein a protein is produced other than by purifying it from cells that naturally express it, the protein is a derivative, by which is meant that the protein comprises additional sequences not related to the protein so long as those sequences do not substantially reduce the biological activity of the protein. One of skill in the art will be aware of, or will readily be able to ascertain, whether a particular polypeptide variant, fragment, or derivative is functional using assays known in the art. For example, the ability of a variant of a transcription factor to convert a source cell to a target cell type can be assessed using the assays as disclose herein in the Examples. Other convenient assays include measuring the ability to activate transcription of a reporter construct containing 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. The term “increasing the amount of” with respect to increasing an amount of a transcription factor, refers to increasing the quantity 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 transcription factor is “increased” in a cell of interest (e.g., a cell into which an expression cassette directing expression of a polynucleotide encoding one or more transcription factors has been introduced) when the quantity of transcription factor is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more relative to a control (e.g., a glial cell into which none of said expression cassettes have been introduced). However, any method of increasing an amount of a transcription factor is contemplated including any method that increases the amount, rate or efficiency of transcription, translation, stability or activity of a transcription factor (or the pre-mRNA or mRNA encoding it).

In particularly preferred embodiments, the method may include use of a CRISPR activation system (CRISPRa), or variations thereof, for activating/increasing the expression of endogenous genes in the source cell and encoding the transcription factors for which an increased amount is desired, so as to facilitate reprogramming. Such methods are well known to a person skilled in the art, such as those published in Fang et al. Molecular therapy. Nucleic Acids, 20 Nov 2018, 14:184-191, incorporated herein by reference.

In addition, down-regulation or interference of a negative regulator of transcription expression, increasing efficiency of existing transcription (e.g. SINEUP) are also considered.

The term "agent" as used herein means any compound or substance such as, but not limited to, a small molecule, nucleic acid, polypeptide, peptide, drug, ion, etc. An "agent" can be any chemical, entity or moiety, including without limitation synthetic and naturally-occurring proteinaceous and non-proteinaceous entities. In some embodiments, an agent is nucleic acid, nucleic acid analogues, proteins, antibodies, peptides, aptamers, oligomer of nucleic acids, amino acids, or carbohydrates including without limitation proteins, oligonucleotides, ribozymes, DNAzymes, glycoproteins, siRNAs, lipoproteins, aptamers, and modifications and combinations thereof etc. A system or set of components, such as a CRISPR activation system, for example as described herein, is also contemplated as an agent. The term “exogenous,” when used in relation to a protein, gene, nucleic acid, or polynucleotide in a cell or organism refers to a protein, gene, nucleic acid, or polynucleotide that has been introduced into the cell or organism by artificial or natural means; or in relation to a cell, refers to a cell that was isolated and subsequently introduced to other cells or to an 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 within the organism or cell. An exogenous cell may be from a different organism, or it may be from the same organism. By way of a non-limiting example, an exogenous nucleic acid is one that is in a chromosomal location different from that of natural cells, or is otherwise flanked by a different nucleic acid sequence than that found in nature. An exogenous nucleic acid may also be extra-chromosomal, such as an episomal vector.

The methods of the invention include high-throughput screening applications. For example, a high-throughput screening assay may be used which comprises any of the assays according to the invention wherein aliquots of a system that allows the product or expression of a transcription factor are exposed to a plurality of candidate agents within different wells of a multi-well plate. Further, a high-throughput screening assay according to the disclosure involves aliquots of a system that allows the product or expression of a transcription factor which are exposed to a plurality of candidate agents in a miniaturized assay system of any kind.

The method of the disclosure may be "miniaturized" in an assay system through any acceptable method of miniaturization, including but not limited to multi-well plates, such as 24, 48, 96 or 384-wells per plate, microchips or slides. The assay may be reduced in size to be conducted on a micro-chip support, advantageously involving smaller amounts of reagent and other materials. Any miniaturization of the process which is conducive to high-throughput screening is within the scope of the invention.

In any method of the invention the target cells can be transferred into the same mammal from which the source cells were obtained. In other words, the source cells used in a method of the invention can be an autologous cell, i.e. , can be obtained from the same individual in which the target cells are to be administered. Alternatively, the target cell can be allogenically transferred into another individual. Preferably, the cell is autologous to the subject in a method of treating or preventing a medical condition in the individual.

As used herein, “culturing” relates to contacting cells with a cell culture medium, typically for a sufficient time and under conditions to allow cell differentiation or proliferation. The term "cell culture medium" (also referred to herein as a "culture medium" or "medium") as referred to herein is a medium for culturing cells containing nutrients that maintain cell viability and support proliferation. The cell culture medium may contain any of the following in an appropriate combination: salt(s), buffer(s), amino acids, glucose or other sugar(s), antibiotics, serum or serum replacement, and other components such as peptide growth factors, etc. Cell culture media ordinarily used for particular cell types are known to those skilled in the art. Exemplary cell culture medium for use in methods of the invention are shown in Table 2.

Table 2. Cell culture media that can be used to culture various cell types, referred to herein as MG cell media and Photoreceptor cell media

A nucleic acid or vector comprising a nucleic acid as described herein may include one or more of the sequences referred to above in Table 1 or a sequence encoding any one or more of the amino acid sequences listed in Table 1.

The term "expression" refers to the cellular processes involved in producing RNA and proteins and as appropriate, secreting proteins, including where applicable, but not limited to, for example, transcription, translation, folding, modification and processing. The term "isolated" or "partially purified" as used herein refers, in the case of a nucleic acid or polypeptide, to a nucleic acid or polypeptide separated from at least one other component (e.g., nucleic acid or polypeptide) that is present with the nucleic acid or polypeptide as found in its natural source and/or that would be present with the nucleic acid or polypeptide when expressed by a cell, or secreted in the case of secreted polypeptides. A chemically synthesized nucleic acid or polypeptide or one synthesized using in vitro transcription/translation is considered "isolated".

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 nucleic acids to which they are linked. Vectors capable of directing the expression of genes to which they are operatively linked are referred to herein as "expression vectors". Thus, an "expression vector" is a specialized vector that contains the necessary regulatory regions needed for expression of a gene of interest in a host cell. In some embodiments the gene of interest is operably linked to another sequence in the vector. Vectors can be viral vectors or non-viral vectors. Should viral vectors be used, it is preferred the viral vectors are replication defective, which can be achieved for example by removing all viral nucleic acids that encode for replication. A replication defective viral vector will still retain its infective properties and enters the cells in a similar manner as a replicating adenoviral vector, however once admitted to the cell a replication defective viral vector does not reproduce or multiply. Vectors also encompass liposomes and nanoparticles and other means to deliver DNA molecule to a cell.

The term "operably linked" means that the regulatory sequences necessary for expression of the coding sequence are placed in the DNA molecule in the appropriate positions relative to the coding sequence so as to effect expression of the coding sequence. This same definition is sometimes applied to the arrangement of coding sequences and transcription control elements (e.g. promoters, enhancers, and termination elements) in an expression vector. The term "operatively linked" includes having an appropriate start 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 sequence, and production of the desired polypeptide encoded by the polynucleotide sequence. The term "viral vectors" refers to the use of viruses, or virus-associated vectors as carriers of a nucleic acid construct into a cell. Constructs may be integrated and packaged into non-replicating, defective viral genomes like Adenovirus, Adeno- associated virus (AAV), or Herpes simplex virus (HSV) or others, including retroviral and lentiviral vectors, for infection or transduction into cells. The vector may or may not be incorporated into the cell's genome. The constructs may include viral sequences for transfection, if desired. Alternatively, the construct may be incorporated into vectors capable of episomal replication, e.g EPV and EBV vectors.

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

As used herein, the term "non-integrating viral vector" refers to a viral vector that does not integrate into the host genome; the expression of the gene delivered by the viral vector is temporary. Since there is little to no integration into the host genome, non integrating viral vectors have the advantage of not producing DNA mutations by inserting at a random point in the genome. For example, a non-integrating viral vector remains extra-chromosomal and does not insert its genes into the host genome, potentially disrupting the expression of endogenous genes. Non-integrating viral vectors can include, but are not limited to, the following: adenovirus, alphavirus, picornavirus, and vaccinia virus. These viral vectors are "non-integrating" viral vectors as the term is used herein, despite the possibility that any of them may, in some rare circumstances, integrate viral nucleic acid into a host cell's genome. What is critical is that the viral vectors used in the methods described herein do not, as a rule or as a primary part of their life cycle under the conditions employed, integrate their nucleic acid into a host cell's genome.

The vectors described herein can be constructed and engineered using methods generally known in the scientific literature to increase their safety for use in therapy, to include selection and enrichment markers, if desired, and to optimize expression of nucleotide sequences contained thereon. The vectors should include structural components that permit the vector to self-replicate in the source cell type. For example, the known Epstein Barr oriP/Nuclear Antigen-1 (EBNA-I) combination (see, e.g., Lindner, S.E. 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 as if set forth herein in its entirety) is sufficient to support vector self-replication and other combinations known to function in mammalian, particularly primate, cells can also be employed. Standard techniques for the construction of expression vectors suitable for use in the present invention are well- known to one of ordinary 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, N. Y. 2001), incorporated herein by reference as if set forth in its entirety.

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

Suitable reprogramming vectors are any described herein, including episomal vectors, such as plasmids, that do not encode all or part of a viral genome sufficient to give rise to an infectious or replication-competent virus, although the vectors can contain structural elements obtained from one or more virus. One or a plurality of 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 transcriptional promoter can provide transcriptional control for a plurality of transgenes, which can be provided as an expression cassette. Separate expression cassettes on a vector can be under the transcriptional control of separate strong, constitutive promoters, which can be copies of the same promoter or can be distinct promoters. Various heterologous promoters are known in the art and can be used depending on factors such as the desired expression level of the transcription factor. It can be advantageous, as exemplified below, to control transcription of separate expression cassettes using distinct promoters having distinct strengths in the source cells. Another consideration in selection of the transcriptional promoters is the rate at which the promoter(s) is silenced. The skilled artisan will appreciate that it can be advantageous to reduce expression of one or more transgenes or transgene expression cassettes after the product of the gene(s) has completed or substantially completed its role in the reprogramming method. Exemplary promoters are the human EF1a elongation factor promoter, CMV cytomegalovirus immediate early promoter and CAG chicken albumin promoter, and corresponding homologous promoters from other species. In human somatic cells, both EF1a and CMV are strong promoters, but the CMV promoter is silenced more efficiently than the EF1a promoter such that expression of transgenes under control of the former is turned off sooner than that of transgenes under control of the latter. The transcription factors can be expressed in the source cells in a relative ratio that can be varied to modulate reprogramming efficiency. Preferably, where a plurality of transgenes is encoded on a single transcript, an internal ribosome entry site is provided upstream of transgene(s) distal from the transcriptional promoter. Although the relative ratio of factors can vary depending upon the factors delivered, one of ordinary skill in possession of this disclosure can determine an optimal ratio of factors.

The skilled artisan will appreciate that the advantageous efficiency of introducing all factors via a single vector rather than via a plurality of vectors, but that as total vector size increases, it becomes increasingly difficult to introduce the vector. The skilled artisan will also appreciate that position of a transcription factor on a vector can affect its temporal expression, and the resulting reprogramming efficiency. As such, Applicants employed various combinations of factors on combinations of vectors. Several such combinations are here shown to support reprogramming.

After introduction of the reprogramming vector(s) and while the source cells are being reprogrammed, the vectors can persist in target cells while the introduced transgenes are transcribed and translated. Transgene expression can be advantageously downregulated or turned off in cells that have been reprogrammed to a target cell type. The reprogramming vector(s) can remain extra-chromosomal. At extremely low efficiency, the vector(s) can integrate into the cells' genome. The examples that follow are intended to illustrate but in no way limit the present invention.

Suitable methods for nucleic acid delivery for transformation of a cell for use with the current invention are believed to include virtually any method by which a nucleic acid (e.g., DNA) can be introduced into a cell as described herein or as would be known to one of ordinary 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 such as by ex vivo transfection (Wilson et al. , Science, 244:1344-1346, 1989, Nabel and Baltimore, Nature 326:711-713, 1987), optionally with a lipid-based transfection reagent 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 incorporated herein by reference), including microinjection (Harland and Weintraub, J. Cell 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); 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., Methods Enzymol., 149:157-176, 1987; Wong et al., Gene, 10:87-94, 1980; Kaneda et al., Science, 243:375-378, 1989; Kato et al., J Biol. Chem., 266:3361-3364, 1991) and receptor-mediated transfection (Wu and Wu, Biochemistry, 27:887-892, 1988; Wu and Wu, J. Biol. Chem., 262:4429-4432, 1987); and any combination of such methods, each of which is incorporated herein by reference.

A number of polypeptides capable of mediating introduction of associated molecules into a cell have been described previously and can be adapted to the present invention. See, e.g., 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), the 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 FGF signal sequence (kFGF); protein transduction domain-4 (PTD4); Penetratin, M918, Transportan-10; a nuclear localization sequence, a PEP-I peptide; an amphipathic peptide (e.g., an MPG peptide); delivery enhancing transporters such as described in U.S. Pat. No. 6,730,293 (including but not limited to an peptide sequence comprising at least 5-25 or more contiguous arginines or 5-25 or more arginines in a contiguous set of 30, 40, or 50 amino acids; including but not limited to an peptide having sufficient, e.g., at least 5, guanidino or amidino moieties); and commercially available Penetratin™ 1 peptide, and the Diatos Peptide Vectors (“DPVs”) of the Vectocell® platform available from Daitos S.A. of Paris, France. See also, WO/2005/084158 and WO/2007/123667 and additional transporters described therein. Not only can these proteins pass through the plasma membrane but the attachment of other proteins, such as the transcription factors described herein, is sufficient to stimulate the cellular uptake of these complexes.

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

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

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

A condition associated with or cause by degeneration, or loss, of photoreceptor cells is any one of retinitis pigmentosa, age-related macular degeneration,.

A disease or disorder or condition associated with, or caused by, degeneration or loss of photoreceptors may be any one of macular degeneration such as age-related macular degeneration, whether at early or late stage, retinitis pigmentosa, choroideremia or diabetic retinopathy. The disease or condition may be wet or dry age- related macular degeneration. The disease or condition may be myopic macular degeneration. The disease or condition may be Stargardt disease. In some instances, the patient has been diagnosed with early or intermediate stage age-related macular degeneration, and/or the cells or cell populations of the invention described herein are administered during such early or intermediate stage. In some embodiments, the disease or condition may be retinitis pigmentosa.

In some embodiments, the loss of photoreceptors is a complete loss of photoreceptors. In some embodiments, the patient has eyesight of 20/60 or worse including 20/80 or worse, 20/100 or worse, 20/120 or worse, 20/140 or worse, 20/160 or worse, 20/180 or worse, 20/200 or worse, 20/400 or worse, 20/800 or worse, or 20/1000 or worse.

Administration of a cell or cell population to an individual in need thereof to treat a condition associated with or cause by degeneration of photoreceptor cells, may be by any method known in the art.

In the methods of the invention, cells to be transplanted are transferred to a recipient in any physiologically acceptable excipient comprising an isotonic excipient prepared under sufficiently sterile conditions for human administration. For general principles in medicinal formulation, 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. Choice of the cellular excipient and any accompanying elements of the composition will be adapted in accordance with the route and device used for administration. The cells may be introduced by injection, catheter, or the like. The cells may be frozen at liquid nitrogen temperatures and stored for long periods of time, being capable of use on thawing. If frozen, the cells will usually be stored in a 10% DMSO, 50% FCS, 40% RPMI 1640 medium.

The pharmaceutical preparations of the invention are optionally packaged in a suitable container with written instructions for a desired purpose. Such formulations may comprise a cocktail of retinal differentiation and/or trophic factors, in a form suitable for combining with cell or cell population of the invention as described herein. Such a composition may further comprise suitable buffers and/or excipients appropriate for transfer into an animal. The cell or cell population of the invention as described herein may be formulated with a pharmaceutically acceptable carrier. For example, cell or cell population of the invention as described herein may be administered alone or as a component of a pharmaceutical formulation. The subject compounds may be formulated for administration in any convenient way for use in medicine. Pharmaceutical preparations suitable for administration may comprise the cell or cell population of the invention as described herein in combination with one or more pharmaceutically acceptable sterile isotonic aqueous or nonaqueous solutions (e.g., balanced salt solution (BSS)), dispersions, suspensions or emulsions, or sterile powders which may be reconstituted into sterile injectable solutions or dispersions just prior to use, which may contain antioxidants, buffers, bacteriostats, solutes or suspending or thickening agents. Exemplary pharmaceutical preparations comprises the cell or cell population of the invention as described herein in combination with ALCON® BSS PLUS® (a balanced salt solution containing, in each mL, sodium chloride 7.14 mg, potassium chloride 0.38 mg, calcium chloride dihydrate 0.154 mg, magnesium chloride hexahydrate 0.2 mg, dibasic sodium phosphate 0.42 mg, sodium bicarbonate 2.1 mg, dextrose 0.92 mg, glutathione disulfide (oxidized glutathione) 0.184 mg, hydrochloric acid and/or sodium hydroxide (to adjust pH to approximately 7.4) in water).

When administered, the pharmaceutical preparations for use in this disclosure may be in a pyrogen-free, physiologically acceptable form.

The preparation comprising a cell or cell population of the invention as described herein used in the methods described herein may be transplanted in a suspension, gel, colloid, slurry, or mixture. Further, the preparation may desirably be encapsulated or injected in a viscous form into the vitreous humor for delivery to the site of retinal or choroidal damage. Also, at the time of injection, cryopreserved cell or cell population of the invention as described herein may be resuspended with commercially available balanced salt solution to achieve the desired osmolality and concentration for administration by subretinal injection. The preparation may be administered to an area of the pericentral macula that was not completely lost to disease, which may promote attachment and/or survival of the administered cells.

The cell or cell population of the invention as described herein may be frozen (cryopreserved) as described herein. Upon thawing, the viability of such cells may 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 about 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 thawing 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 cell number initially frozen are harvested in a viable state after thawing). In some instances, the viability of the cells prior to and after thawing is about 80%. In some instances, at least 90% or at least 95% or about 95% of cells that are frozen are recovered. The cells may be frozen as single cells or as aggregates.

The cell or cell population of the invention as described herein may be delivered in a pharmaceutically acceptable ophthalmic formulation by intraocular injection. When administering the formulation by intravitreal injection, for example, the solution may be concentrated so that minimized volumes may be delivered. Concentrations for injections may be at any amount that is effective and non-toxic, depending upon the factors described herein. The pharmaceutical preparations of cell or cell population of the invention as described herein for treatment of a patient may be formulated at doses of at least about 104 cells/mL. The cell or cell population of the invention as described herein preparations for treatment of a patient are formulated at doses of at least about 10³, 10⁴, 10^s, 10⁶, 10⁷, 10^s, 10⁹, or 10¹⁰ cells/mL.

The pharmaceutical preparations of cells of the invention described herein may comprise at least about 1 ,000; 2,000; 3,000; 4,000; 5,000; 6,000; 7,000; 8,000; or 9,000 photoreceptor cells. The pharmaceutical preparations of photoreceptor cells may comprise at least about 1 ^c10⁴, 2^c10⁴, 3^c10⁴, 4^c10⁴, 5^c10⁴, 6^c10⁴, 7^c10⁴, 8^c10⁴, 9^c10⁴,

1x10⁵, 2^c10⁵, 3^c10⁵, 4^c10⁵, 5^c10⁵, 6^c10⁵, 7^c10⁵, 8^c10⁵, 9^c10⁵, 1^c10⁶, 2^c10⁶, 3^c10⁶,

4^c10⁶, 5^c10⁶, 6^c10⁶, 7^c10⁶, 8^c10⁶, 9^c10⁶, 1 ^c10⁷, 2^c10⁷, 3^c10⁷, 4^c10⁷, 5^c10⁷, 6^c10⁷,

7^c10⁷, 8^c10⁷, 9^c10⁷, 1^c10⁸, 2^c10⁸, 3^c10⁸, 4^c10⁸, 5^c10⁸, 6^c10⁸, 7^c10⁸, 8^c10⁸, 9^c10⁸,

1^c10⁹, 2^c10⁹, 3^c10⁹, 4^c10⁹, 5^c10⁹, 6^c10⁹, 7^c10⁹, 8^c10⁹, 9^c10⁹, 1^c10¹⁰, 2^c10¹⁰, 3^c10¹⁰, 4^c10¹⁰, 5^c10¹⁰, 6^c10¹⁰, 7^c10¹⁰, 8^c10¹⁰, or 9^c10¹⁰ photoreceptor cells. The pharmaceutical preparations of photoreceptor cells may comprise at least about 1 x102- 1x10³, 1x10²-1x10⁴, 1x10⁴-1 x10⁵, or 1 ^c10³-1^c10⁶ photoreceptor cells. The pharmaceutical preparations of 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 photoreceptor cells. For example, the pharmaceutical preparation of photoreceptor cells may comprise at least about 20,000-200,000 photoreceptor cells in a volume at least about 50-200 mI_. Further, the pharmaceutical preparation of photoreceptor cells may comprise about 50,000 photoreceptor is in a volume of 150 mI_, about 200,000 photoreceptor cells in a volume of 150 mI_, or at least about 180,000 photoreceptor cells in a volume at least about 150 mI_.

In the aforesaid pharmaceutical preparations and compositions, the number of photoreceptor cells or concentration of photoreceptor cells may be determined by counting viable cells and excluding non-viable cells. For example, non-viable photoreceptor may be detected by failure to exclude a vital dye (such as Trypan Blue), or using a functional assay (such as the ability to adhere to a culture substrate, phagocytosis, etc.). Additionally, the number of photoreceptor cells or concentration of photoreceptor cells may be determined by counting cells that express one or more photoreceptor cell markers and/or excluding cells that express one or more markers indicative of a cell type other than photoreceptor.

The photoreceptor cells may be formulated for delivery in a pharmaceutically acceptable ophthalmic vehicle, such that the preparation is maintained in contact with the ocular surface for a sufficient time period to allow the cells to penetrate the affected regions of the eye, as for example, the anterior chamber, posterior chamber, vitreous body, aqueous humor, vitreous humor, cornea, iris/ciliary, lens, choroid, retina (e.g. sub retina), sclera, suprachoridal space, conjunctiva, subconjunctival space, episcleral space, intracorneal space, epicorneal space, pars plana, surgically-induced avascular regions, or the macula.

The methods described herein may further comprise the step of monitoring the efficacy of treatment or prevention by measuring electroretinogram responses, optomotor acuity threshold, or luminance threshold in the subject. The method may also comprise monitoring the efficacy of treatment or prevention by monitoring immunogenicity of the cells or migration of the cells in the eye.

Note, that the human cells may be used in human patients, as well as in animal models or animal patients. For example, the human cells may be tested in mouse, rat, cat, dog, or non-human primate models of retinal degeneration. Additionally, the human cells may be used therapeutically to treat animals in need thereof, such as in veterinary medicine. Examples of veterinary subjects or patients include without limitation dogs, cats, and other companion animals, and economically valuable animals such as livestock and horses.

Examples

The inventor(s) optimized a CRISPRa system in mammalian cells as previously described (Fang et al. Molecular therapy. Nucleic Acids, 20 Nov 2018, 14:184-191), which allowed the inventor(s) to activate efficient expression of up to 9 genes simultaneously. This platform greatly enhanced the capacity to perform in vitro screening in human Muller glial cell line (MIO-M1) to identify various transcription factors or combinations that promote reprogramming into iPH (induced photoreceptors). Validation of the iPH quality was performed by photoreceptor marker analysis using qPCR and immunocytochemistry, as well as single cell transcriptomic using the 10x Chromium system and Hiseq 2500 next generation sequencing.

Example 1

The loss of photoreceptors is a key hallmark of many incurable blinding diseases and regenerative medicine has great potentials of alleviating blindness in patients. Here the inventor(s) describe the identification of transcription factors that promote reprograming of human Muller glial cells into photoreceptors (termed induced photoreceptors, iPH) in vitro.

The inventor(s) adapted the CRISPR activation (CRISPRa) system to activate expression of endogenous genes, which allowed them to activate up to 9 transcription factors simultaneously (specifically, the individual transcription factors or transcription factor sets as shown in Table 4). Using this CRISPRa platform, the inventor(s) have screened and identified individual transcription factors and transcription factor combinations or cocktails that promote reprogramming of human Muller glia into iPH in vitro. qPCR and immunocytochemical analysis demonstrated that the reprogrammed iPH expressed a panel of photoreceptor markers, including RHO and PDE6B. Using multi-electrode array analysis, it was also demonstrated that the iPH possess functional electrophysiology. To comprehensively analyse the iPH, the inventor(s) performed single cell transcriptome profiling of iPH cells. Transcriptome analysis demonstrated the transition of glial to neuron through reprogramming, the activation of photoreceptor markers in iPH and the presence of different reprogramming stages. Using single cell transcriptomics, the iPH were also benchmarked against a human adult retina gene atlas. The results showed that iPH reprogramming promoted transcriptome transitions from Muller glia to photoreceptors, supporting the quality of the derived iPH.

The study described herein demonstrated the use of direct reprogramming to convert human Muller glia into photoreceptors, providing a potential cell source for tissue engineering and regenerative medicine. Compared to the use of induced pluripotent stem cells, a major advantage of the direct reprogramming approach is that it bypasses the procedure of resetting somatic cells into a pluripotent stage, thus reducing the time needed to derive photoreceptors in vitro which in turn reduces the cost of cell production

Materials and methods

Reprograming to generate iPH cells

The CRISPRa system is utilised to promote reprogramming of human MG cells to iPH cells, either by Lipofectamine transfection or lentiviral transduction. For Lipofectamine transfection, sgRNA expression cassettes are generated as described by Fang et al. supra. Human MG cells (MIO-M1) cultured in MG Cell Media were transfected with 40 ng sgRNA expression cassette and 800 ng Sp-dCas9VPR plasmid (Addgene) per well in 12 well plate format using Lipofectamine 3000 following manufacturer’s instructions. From day 3 onwards, the media is switched to the Photoreceptor Cell Media and treated with 10ng/ml Trichostain A. The transfection is repeated on day 5 and day 10 to allow prolonged expression of CRISPRa system and the sample is analysed on day 14 for presence of iPH.

For lentiviral transduction, human MG cells cultured in MG Cell Media were first transduced with lentiviruses carrying the CRISPRa SunTag system (pppHRdSV40- dCas9-10xGCN4_v4-P2A-BFP, pHRdSV40-scFv-GCN4-sfGFP-VP64-GB1-NLS) on day -1, followed by transduction of lentiviruses carrying specific sgRNA (LentiGuide- sgRNA-puro) on day 0. 8 pg/ml of polybrene (Sigma) was added to improve transduction efficiency. From day 3 onwards, the media is switched to the Photoreceptor Cell Media and treated with 10 ng/ml Trichostain A and the sample is analysed on day 14 for presence of iPH. qPCR analysis

Total RNA were extracted using the lllustra RNAspin kit and treated with DNase 1(GE Healthcare). cDNA synthesis and Taqman qPCR were performed as previously described (Hung et al. 2016; Aging 8 (5): 945-57), using the following Taqman probes for RHO (Hs00892431_m1) and the housekeeping gene b-actin (Hs99999903_m1). The Taqman assay was processed in an ABI 7500 or StepOne plus (Thermo Fisher) and gene expression was analysed using the AACt method.

Immunocvtochemistrv

Standard immunocytochemistry procedures were carried out as previously described (Wong et al. 2011, Stem Cells, 29(10), 1517-1527). In brief, samples were fixed in 4% paraformaldehyde, followed by blocking and permeabilization (0.1% Triton X-100). The samples were then immunostained with antibodies against RHO (Abeam, #AB5417) or PDE6B (Abeam, AB5663), followed by the appropriate Alexa Fluor 488 or Alex Fluor 568 secondary antibodies (Thermo Fisher or Abeam), and nuclear counterstain with DAPI (Sigma). Samples were imaged using a Zeiss Axio Vert.AI fluorescent microscope or Nikon Eclipse TE2000-U.

Electrophvsioloqical analysis using microelectrode array

The microelectrode array (MEA) recording system (Multichannel Systems, Reutlingen, Germany) was used to measure the electrophysiological response of iPH. The MG cells were reprogrammed on MEA plates and recordings were performed after 14 days after that. Data were analyzed with MC Rack software.

Single cell RNA sequencing

Human MG cells and iPHs were dissociated into single cells using 0.25% Trypsin- EDTA and filtered using 30pm MACS Smart Strainer (Miltenyi). The single cells were captured using the 10X Chromium system (10X Genomics) and barcoded cDNA libraries were prepared using the Single cell 3’ mRNA kit, followed by 100bp paired-end sequencing using the lllumina Hi-Seq2500 (Australian Genome Research Facility). Bioinformatic analysis and quality control was performed using the 10X Cellranger pipeline and Seurat package as described in Lukowski et al. 2019, EMBO Journal, 38(18); e100811.

Table 3: Guide RNAs used to increase expression of transcription factors.

Results

Figure 1A shows the fold change gene expression following CRISPR activation to induce expression of 9 candidate transcription factors in human MG cells (MIOMI) using single sgRNA, or multiplex expression of 9 sgRNA. n = 3, error bars = SEM. B.RHO mRNA expression (fold change relative to mock) is shown for 125 reprogramming experiments to identify transcription factor cocktails that reprogram human MG cells into induced photoreceptor cells (iPH) (Arrows indicate: ANNr = ASCL1+NEUROD1+NRL; ANrR = ASCL1+NRL+RAX; 9 genes = ASCL1+NEUROD1+CRX+OTX2+NRL+RAX+ RORB+NR2E3PAX6). Results represent mean of technical triplicates ± SEM. C. RHO mRNA expression (fold change relative to mock) for selected reprogramming conditions as listed in Table 4.

Figures 2A and B show fluorescence microscopy images of derived iPH produced according to the invention. Staining with RHO and DAPI indicates that the iPH are positive for rod marker RHO (Figure 2A). Staining with rod marker PDE6B also indicates that the derived iPH are positive for this marker (Figure 2B). Figure 2 C provides a violin plot of multielectrode array of mock control and iPH. Pre-illumination and post-illumination (left- and right-hand sides, respectively, for mock and iPH samples), demonstrate that the iPH display functional electrophysiological responses following light stimulation.

Results of single cell RNAseq indicate the presence of an iPH subpopulation (arrow) in which all 9 rod markers are upregulated. C1 - C8 represent clusters identified by single cell transcriptomics and represent subpopulations within the reprogrammed culture. (Figure 3A). Principal component analysis of transcriptomics is expected to induce a transcriptional shift from MG cells to rods (Figure 3B). Using a single cell human retinal gene atlas as a benchmark, the pilot scRNAseq data captured various reprogramming stages of iPH from MG cells to rod photoreceptors specifically (marked line). (Figure 3C).

Table 4: Summary of individual or combinations of transcription factors - abbreviations shown in Table 3 - and the mean RHO expression as fold change relative to mock of resulting induced photoreceptor cells.

Claims

1. An in vitro method for reprogramming a source cell, the method comprising increasing the protein expression of one or more transcription factors, or variant thereof, in the source cell, wherein the source cell is reprogrammed to exhibit at least one characteristic of a target cell, wherein:

- the source cell is a glial cell;

- the target cell is a photoreceptor cell; and

2. An in vitro method of generating a cell exhibiting at least one characteristic of a photoreceptor cell from a source cell, the method comprising:

- the source cell is a glial cell,

3. An in vitro method for reprogramming a source cell to a cell that exhibits at least one characteristic of a photoreceptor cell, the method comprising:

- providing a source cell, or a cell population comprising a source cell;

- transfecting said source cell with one or more nucleic acids comprising a nucleotide sequence that encodes one or more transcription factors; and

- culturing said cell or cell population, and optionally monitoring the cell or cell population for at least one characteristic of a photoreceptor cell, wherein: the source cell is a glial cell and

4. An in vitro method for reprogramming a source cell to a cell that exhibits at least one characteristic of a photoreceptor cell, the method comprising:

- providing a source cell, or a cell population comprising a source cell;

- the source cell is a glial cell and

5. A method of claim 4, wherein the one or more nucleic acids comprise sgRNAs for use in a CRISPR activation system for increasing expression of genes encoding the one or more transcription factors.

6. A method of any one of claims 3 to 5, wherein the method comprises transfecting the source cell with nucleic acids encoding or for increasing the expression of least two of: ASCL1, NEUROD1, NRL, NR2E3, RAX, RORB, OTX2, CRX and PAX6; at least three of ASCL1, NEUROD1, NRL, NR2E3, RAX, RORB, OTX2, CRX and PAX6; at least four of ASCL1, NEUROD1, NRL, NR2E3, RAX, RORB, OTX2, CRX and PAX6; at least five of ASCL1, NEUROD1, NRL, NR2E3, RAX, RORB, OTX2, CRX and

PAX6; at least six of ASCL1, NEUROD1, NRL, NR2E3, RAX, RORB, OTX2, CRX and

PAX6; at least 7 of ASCL1, NEUROD1, NRL, NR2E3, RAX, RORB, OTX2, CRX and

PAX6; at least 8 of ASCL1, NEUROD1, NRL, NR2E3, RAX, RORB, OTX2, CRX and

PAX6; or all 9 of ASCL1, NEUROD1, NRL, NR2E3, RAX, RORB, OTX2, CRX and PAX6.

7. A method of any one of claims 1 to 5, wherein the transcription factors are one or more of those selected from ASCL1, NEUROD1, NRL, NR2E3, RAX, RORB, OTX2, CRX and PAX6, wherein the combination results in a photoreceptor, or photoreceptor-like, cell with a fold increase in RHO mRNA expression of equal to, or greater than, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70 or 75 fold compared to the RHO expression in the source cell type.

8. A method of claim 7, wherein the fold increase is equal to, or greater than, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70 or 75 fold compared to the RHO expression in the source cell type.

9. A method of claim 8, wherein the fold increase is equal to, or greater than, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70 or 75 fold compared to the RHO expression in the source cell type.

10. A method of any one of claims 1 to 9, wherein the source cell is a Muller glial cell, and the transcription factors, or variants thereof, are:

(a) ASCL1 , NEUROD1 , NRL, NR2E3, RAX, RORB, OTX2, CRX and PAX6;

(b) ASCL1, NEUROD1 and NRL;

(c) ASCL1 , NRL and RAX;

(d) ASCL1, NRL and CRX;

(e) ASCL1 , NRL, RAX and CRX;

(f) ASCL1 , NRL and OTX2;

(g) ASCL1 , NRL and NR2E3;

(h) ASCL1 , NRL and RORB;

(i) RORB, OTX2, CRX and NRL;

0 ASCL1, NRL, RORB and NR2E3;

(k) ASCL1 , NRL, CRX and OTX2; (L) ASCL1 , NEUROD1 , NRL and CRX;

(m) ASCL1 , NEUR0D1 , NRL and NR2E3;

(n) NR2E3, 0TX2, RAX and NEUR0D1 ;

(o) OTX2;

(p) ASCL1 , NRL, NR2E3 and CRX;

(q) ASCL1 , NEUROD1 , NRL and RORB;

(r) ASCL1 , NEUROD1 , NRL and OTX2;

(s) RAX;

(t) OTX2, RAX and NEUROD1;

(u) ASCL1 , NRL, NR2E3 and RAX;

(v) RORB;

(w) NR2E3, OTX2, CRX, RAX and NEUROD1 ;

(x) ASCL1 ;

(y) NEUROD1;

(z) PAX6;

(aa) NEUROD1, CRX, NR2E3 and RAX;

(bb) CRX;

(cc) ASCL1, NRL, RORB and OTX2;

(dd) NRL

(ee) ASCL1 , NEUROD1 , NRL, RAX and NR2E3; (ff) ASCL1, NEUROD1, NRL, NR2E3 and CRX; (gg) ASCL1 , NRL, RORB and CRX; (hh) NR2E3;

(ii) CRX, RAX and NEUROD1 ; or

(jj) CRX, OTX2 and NRL.

11. A method of any one of claims 1 to 9, wherein the source cell is a Muller glial cell, and the transcription factors, or variants thereof, are:

(a) ASCL1 , NEUROD1 , NRL, NR2E3, RAX, RORB, OTX2, CRX and PAX6;

(b) ASCL1, NEUROD1 and NRL;

(c) ASCL1 , NRL and RAX;

(d) ASCL1, NRL and CRX;

(e) ASCL1 , NRL, RAX and CRX;

(f) ASCL1 , NRL and OTX2;

(g) ASCL1 , NRL and NR2E3;

(h) ASCL1 , NRL and RORB;

(i) RORB, OTX2, CRX and NRL;

0 ASCL1, NRL, RORB and NR2E3;

(k) ASCL1 , NRL, CRX and OTX2;

(L) ASCL1 , NEUROD1 , NRL and CRX;

(m) ASCL1 , NEUROD1 , NRL and NR2E3;

(n) NR2E3, OTX2, RAX and NEUROD1 ;

(o) OTX2;

(p) ASCL1 , NRL, NR2E3 and CRX; (q) ASCL1 , NEUROD1 , NRL and RORB;

(r) ASCL1 , NEUROD1 , NRL and OTX2;

(s) RAX;

(t) OTX2, RAX and NEUROD1;

(u) ASCL1 , NRL, NR2E3 and RAX;

(v) RORB; or

(w) NR2E3, OTX2, CRX, RAX and NEUROD1.

12. A method of any one of claims 1 to 9, wherein the source cell is a Muller glial cell, and the transcription factors, or variants thereof, are:

(a) ASCL1 , NEUROD1 , NRL, NR2E3, RAX, RORB, OTX2, CRX and PAX6;

(b) ASCL1, NEUROD1 and NRL;

(c) ASCL1 , NRL and RAX;

(d) ASCL1, NRL and CRX;

(e) ASCL1 , NRL, RAX and CRX;

(f) ASCL1 , NRL and OTX2;

(g) ASCL1 , NRL and NR2E3;

(h) ASCL1 , NRL and RORB;

(i) RORB, OTX2, CRX and NRL;

0 ASCL1, NRL, RORB and NR2E3;

(k) ASCL1 , NRL, CRX and OTX2;

(L) ASCL1 , NEUROD1 , NRL and CRX; or

(m) ASCL1 , NEUROD1 , NRL and NR2E3.

13. A method of any one of claims 1 to 9, wherein the source cell is a Muller glial cell, and the transcription factors, or variants thereof, are:

(a) ASCL1 , NEUROD1 , NRL, NR2E3, RAX, RORB, OTX2, CRX and PAX6;

(b) ASCL1, NEUROD1 and NRL; or

(c) ASCL1, NRL and RAX.

14. A method of any one of claims 1 to 9, wherein the source cell is a Muller glial cell, and the transcription factors, or variants thereof, are ASCLI, NEUROD1, NRL, NR2E3, RAX, RORB, OTX2, CRX and PAX6.

15. A method of claim 3, wherein the source cell is a Muller glial cell, and the transcription factors, or variants thereof, are:

(a) ASCL1 , NEUROD1 , NRL, NR2E3, RAX, RORB, OTX2, CRX and PAX6;

(b) ASCL1, NEUROD1 and NRL; or

(c) ASCL1, NRL and RAX.

16. A method of claim 3, wherein the source cell is a Muller glial cell, and the transcription factors, or variants thereof, are ASCL1, NEUROD1, NRL, NR2E3, RAX, RORB, OTX2, CRX and PAX6.

17. A method of claim 5, wherein the source cell is a Muller glial cell, and the transcription factors, or variants thereof, are:

(a) ASCL1 , NEUROD1 , NRL, NR2E3, RAX, RORB, OTX2, CRX and PAX6;

(b) ASCL1, NEUROD1 and NRL; or

(c) ASCL1, NRL and RAX.

18. A method of claim 5, wherein the source cell is a Muller glial cell, and the transcription factors, or variants thereof, are ASCLI, NEUROD1, NRL, NR2E3, RAX, RORB, OTX2, CRX and PAX6.

19. A method of any one of claims 1 to 18, wherein the nucleic acid encoding the one or more transcription factors is listed in Table 1.

20. A method of any one of claims 1 to 20, wherein the source cell is a human cell.

21. A method of any one of claims 1 to 20, wherein the glial cell is selected from the group consisting of a Muller glial (MG) cell, an astrocyte and a microglia

22. A method of claim 21 , wherein the source cell is a Muller glial cell.

23. A method of any one of claims 2 to 6, wherein culturing the source cell for a sufficient time and under conditions to allow differentiation to a photoreceptor cell including culturing the cells for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 days in a relevant medium as shown in Table 2.

24. A method of any one of claims 1 to 23, wherein the method further comprises the step of expanding the cells exhibiting at least one characteristic of a photoreceptor cell to increase the proportion of cells in the population exhibiting at least one characteristic of a photoreceptor cell.

25. A method of any one of claims 1 to 24, wherein the method further comprises the step of administering the cells, or cell population including a cell, exhibiting at least one characteristic of a target cell type, to an individual.

26. A cell exhibiting at least one characteristic of a photoreceptor cell produced by a method of any one of claims 1 to 25.

27. The cell of claim 26, wherein the cell comprises at least one characteristic of a rod cell.

28. A population of cells, wherein at least 1% of cells exhibit at least one characteristic of a photoreceptor cell and those cells are produced by a method of any one of claims 1 to 25.

29. A population of cells of claim 27, wherein at least 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 a photoreceptor cell.

30. A kit for producing a cell exhibiting at least one characteristic of a photoreceptor cell, the kit comprising one or more nucleic acids having one or more nucleic acid sequences encoding a transcription factor selected from ASCL1, NEUROD1, NRL, NR2E3, RAX, RORB, OTX2, CRX and PAX6.

31. A kit for producing a cell exhibiting at least one characteristic of a photoreceptor cell, the kit comprising one or more nucleic acids, optionally sgRNAs, for increasing the expression of one or more genes encoding a transcription factor selected from ASCL1, NEUROD1, NRL, NR2E3, RAX, RORB, OTX2, CRX and PAX6.

32. A kit of claim 30 or 31 , wherein the transcription factors are one or more of the specific combinations defined in (a) to (jj) of claim 10.

33. A kit of any one of claims 30 to 32, wherein the kit further comprises instructions for reprogramming a source cell to a cell exhibiting at least one characteristic of a photoreceptor cell according to any one of the methods of claim 1 to 25.

34. A kit of any one of claims 30 to 33, when used in a method of any one of claims 1 to 25.

35. A method of treating a condition associated with or caused by degeneration of photoreceptor cells in an individual in need thereof, the method comprising administering to the individual a cell of claim 26 or 27 or cell population of claim 28 or 29.

36. Use of a cell of claim 26 or 27 or cell population of claim 28 or 29 in the manufacture of a medicament for the treatment of a condition associated with or caused by degeneration of photoreceptor cells in an individual in need thereof.

37. A cell of claim 26 or 27 or cell population of claim 28 or 29, for use in the treatment of a condition associated with or caused by degeneration of photoreceptor cells in an individual in need thereof.

38. A method of claim 35, use of claim 36, or cell or cell population of claim 37, wherein the condition associated with or cause by degeneration of photoreceptor cells is any one of retinitis pigmentosa, age-related macular degeneration, choroideremia and diabetic retinopathy.

39. A nucleic acid comprising a nucleotide sequence encoding one or more of the transcription factors defined in any one of claims 1 to 18.

40. A nucleic acid comprising a nucleotide sequence encoding one or more of the sets of transcription factors defined in claim 10.

41. A vector comprising a nucleic acid of claim 39 or 40.

42. An in vitro or ex vivo cell comprising a nucleic acid of claim 39 or 40, or vector of claim 41.

43. A CRISPR activation system for increasing the expression one or more sets of transcription factors defined in claim 10

44. A CRISPR activation system of claim 43 comprising the sgRNAs described in Table 3.