WO2015077498A1

WO2015077498A1 - Methods and compositions for treating disorders of the eye

Info

Publication number: WO2015077498A1
Application number: PCT/US2014/066696
Authority: WO
Inventors: Sai Chavala; Aiguo NI
Original assignee: The University Of North Carolina At Chapel Hill
Priority date: 2013-11-20
Filing date: 2014-11-20
Publication date: 2015-05-28

Abstract

The present invention provides methods and compositions for producing induced retinal progenitor cells, induced retinal pigment epithelium cells and induced photoreceptor cells for treatment of disorders of the eye.

Description

METHODS AND COMPOSITIONS FOR TREATING DISORDERS OF THE EYE

STATEMENT OF PRIORITY

This application claims the benefit, under 35 U.S. C. § 119(e), of U.S. Provisional Application Serial No. 61/906,750, filed November 20, 2013, the entire contents of which are incorporated by reference herein.

STATEMENT OF GOVERNMENT SUPPORT

This invention was made with government support under Grant No. K08-EY021 171 awarded by the National Institutes of Health. The government has certain rights in the invention.

FIELD OF THE INVENTION

BACKGROUND OF THE INVENTION

Age-related macular degeneration (ARMD) is the most common cause of severe, irreversible vision loss in Western countries. Non-neovascular "dry" or atrophic ARMD results in vision loss from poorly functioning photoreceptors as a consequence of retinal pigment epithelium (RPE) atrophy. The ability to replace damaged RPE with human embryonic stem cell- (ESC) or induced pluripotent stem cell (iPSC)-derived RPE is being evaluated in clinical trials. However, using cells derived from either of these pluripotent cell sources raises significant concerns; (1) iPSC line heterogeneity leads to variability in the quality and ontogenetic stage of derived RPE-like cells and (2) retinal-like cells derived from pluripotent sources are subject to both contamination from tumor-forming progenitor cells and spontaneous de-differentiation. There is a need to develop direct reprogramming strategies if autologous retinal cell transplantation is to succeed clinically. Compared to iPSCs and ESCs, direct RPE induction has the potential advantages of improved stability through directed RPE epigenome and genome modification, less risk of immune rejection, improved efficiency and quality through RPE specification, and reduced tumor formation risk by avoiding pluripotent cell contamination. The present invention addresses previous shortcomings in the art by providing methods and compositions for producing induced retinal pigment epithelial cells in vitro and in vivo to treat macular degeneration and other ocular disorders. SUMMARY OF THE INVENTION

In one aspect, the present invention provides a method of generating an induced retinal pigment epithelium cell, comprising introducing into a somatic cell one or more transcription factors selected from the group consisting of OTX2, FOXG1, Pax6, Smad6, Lhx2, HNF4a, P NOX2, Sox9, FoxDl, MITF, Mir200, Mir204, Mir211, Crx, Six3, Klf4, C- Myc, Klf9, RARa, Pax2, Smad3, Sox4, Soxl l, Otxl, Pitx2, Nr2fl, Nr2f2₅ Tfec and any combination thereof.

In a further aspect, the present invention provides a method of generating an induced photoreceptor cell, comprising introducing into a somatic cell one or more transcription factors selected from the group consisting of Ascll, Brn2, Rax, Crx, Nrl, NeuroDl, Otx2, Mirl24, FoxGl, Pax6, Ars2, Sox2, Nr2e3, Ror-beta, Blimpl, Mir200, CBP, p300, TR$2, PIAS3, Nr2el, Nrldl, Vsx2, Six6, Rbl, Mirl82, Mirl83, Pax7 and any combination thereof.

Also provided herein is a method of generating an induced retinal progenitor cell, comprising introducing into a somatic cell one or more transcription factors selected from the group consisting of Otx2_} Hesl, c-Myc, Six3, Pax6, Rax, Ascll, Crx, Sox2, Vsx2_s Ikzfl and any combination thereof.

In an additional aspect, the present invention provides a method of generating an induced retinal pigment epithelium cell, comprising introducing into a somatic cell the transcription factor OTX2 plus a cyclic AMP agonist molecule.

Furthermore, the present invention provides a method of generating an induced retinal pigment epithelium cell, comprising introducing into a somatic cell the transcription factors OTX2 and Sox9.

A method is also provided herein of generating an induced retinal pigment epithelium cell, comprising introducing into a somatic cell the transcription factors OTX2 and MITF. In a further aspect, the present invention provides a method of generating an induced retinal pigment epithelium cell, comprising introducing into a somatic cell the transcription factors OTX2, MITF and Sox9. These methods can further comprise introducing into the somatic cell the transcription factor FoxDl and/or introducing into the somatic cell the transcription factor PKNOX2 and/or introducing into the somatic cell the transcription factors Mir200, Mir204 and Mir211 and/or introducing into the somatic cell the transcription factor c-myc and/or introducing into the somatic cell the transcription factor Klf-4.

The methods described herein can further comprise introducing into the somatic cell the transcription factor HNF4a and/or further comprise introducing into the somatic cell one or more transcription factors selected from the group consisting of FOXG1 , Smad6, Lhx2, HNF4a and any combination thereof and/or further comprise introducing into the somatic cell one or more transcription factors selected from the group consisting of Klf4, c-myc, Klf9, RARa, Pax2, Smad3, Sox4, Soxl 1, Otxl, Pitx2, Ni2fl, Nr2f2, Tfec and any combination thereof.

In an additional aspect, the present invention provides a method of generating an induced photoreceptor cell, comprising introducing into a somatic cell one or more transcription factors selected from the group consisting of Ascll, Crx, Nrl, NeuroDl, Rax, Otx2, Mir 124, FoxGl, Pax6, Ars2 and any combination thereof. This method can further comprise introducing into the somatic cell one or more transcription factors selected from the group consisting of Nr2e3, Ror-beta, Blimp 1 , Mir200, CBP, p300, TRp2, PI AS 3, Nr2el , Nrldl, Vsx2, Six6, RBI, Mirl82, Mirl 83, Pax7 and any combination thereof.

In another aspect of this invention, a method is provided of generating an induced retinal progenitor cell, comprising introducing into a somatic cell the transcription factors mir200, mir204 and mir211. This method can further comprise introducing into the somatic cell the transcription factor MITF. These preceding methods can further comprise

introducing into the somatic cell the transcription factor Smad6. These preceding methods can further comprise Introducing into the somatic cell the transcription factors HNF4alpha, Sox9, PKNOX2, Pax6, FoxDl, FoxGl and Otx2. These preceding methods can further comprise introducing into the somatic cell the transcription factor Lhx2. These preceding methods can further comprise introducing into the somatic cell a transcription factor selected from the group consisting of Sox2, Ikzfl, Ascll and any combination thereof.

The present invention additionally provides an induced retinal pigment epithelium cell, an induced photoreceptor cell and/or an induced retinal progenitor cell produced by the respective methods of this invention.

Additional aspects of this invention provide method claims, including, in one embodiment, a method of treating a disorder of the eye in a subject in need thereof, comprising delivering to the eye(s) of the subject an effective amount of a cell of this invention. Also provided herein is a method of reprogramming a cell in the eye of a subject into an induced retinal pigment epithelium cell, comprising introducing into the cell of the eye one or more transcription factors selected from the group consisting of OTX2, FOXG1, Pax6, Smad6, Lhx2, HNF4a, PKNOX2, Sox9, FoxDl, MITF, Mir200, ir204, Mir211, Klf4, c- myc, Klf9, RARa, Pax2, Smad3, Sox4, Soxl 1, Otxl, Pitx2, Nr2fl, Nr2f2, Tfec and any combination thereof.

Further provided herein is a method of reprogramming a cell in the eye of a subject into an induced photoreceptor cell, comprising introducing into the cell of the eye one or more transcription factors selected from the group consisting of Ascll, Crx, Nrl, NeuroDl, Rax, Otx2, Mirl24, FoxGl, Pax6, Ars2, Nr2e3, Ror-beta, Blimpl, Mir200, CBP, p300,

TRp2, PIAS3, Nr2el, Nrldl, Vsx2, Six6, RBI, Mirl82, Mirl83, Pax7 and any combination thereof.

Another aspect of this invention provides a method of reprogramming a cell in the eye of a subject into an induced retinal progenitor cell, comprising introducing into the cell of the eye one or more transcription factors selected from the group consisting of Otx2, Hesl , c- Myc, Six3, Pax6, Rax, Ascll, Crx, Sox2, Vsx2 and any combination thereof,

Additionally provided herein is a method of reprogramming a cell in the eye of a subject into an induced retinal pigment epithelium cell, comprising introducing into the cell of the eye the transcription factors OTX2, Sox9_} FoxDl and MITF. This method can further comprise the steps of introducing into the cell the transcription factor PK OX2. The preceding methods can further comprise introducing into the cell the transcription factors Mir200, Mir204 and Mir211. The preceding methods can further comprise introducing into the cell one or more transcription factors selected from the group consisting of FOXG1, Smad6, Lhx2, HNF4a and any combination thereof. The preceding methods can further comprising introducing into the cell one or more transcription factors selected from the group consisting of Klf4, c-myc, Klf , RARa, Pax2, Smad3, Sox4, Soxl 1, Otxl, Pitx2, Nr2fl, Nr2£2, Tfec and any combination thereof.

Further aspects of this invention include a method of reprogramming a cell in the eye of a subject into an induced photoreceptor cell, comprising introducing into the cell of the eye one or more transcription factors selected from the group consisting of Ascll, Crx, Nrl,

NeuroDl, Rax, Otx2, Mir 124, FoxGl, Pax6, Ars2 and any combination thereof. This method can further comprise introducing into the cell one or more transcription factors selected from the group consisting of Nr2e3, Ror-beta, Blimpl, Mir200, CBP, p300_} TRp2, PIAS3, Nr2el, Nrldl, Vsx2, Six6, RBI, M 82, Mirl83, Pax7 and any combination thereof. Additionally provided herein is a method of reprogramming a cell in the eye of a subject into an induced retinal progenitor cell, comprising introducing into the cell of the eye the transcription factors mir200, mir204 and mir211. This method can further comprise introducing into the cell the transcription factor MITF. The preceding methods can further comprise introducing into the cell the transcription factor Smad6. These preceding methods can further comprise introducing into the cell the transcription factors HNF4alpha, Sox9, PKNOX2, Pax6, FoxDl, FoxGl and Otx2. These preceding methods can further comprise introducing into the cell the transcription factor Lhx2, These preceding methods can further comprise introducing into the cell a transcription factor selected from the group consisting of Sox2, Ikzfl , Ascll and any combination thereof.

Also provided herein is a method of reprogramming a preexisting retinal pigment epithelium (RPE) cell having no function or reduced function into a RPE cell having normal function or increased function, comprising introducing into the preexisting RPE cell one or more transcription factors selected from the group consisting of OTX2, FOXG1, Pax6, Smad6, Lhx2, HNF4ct, PKNOX2, Sox9, FoxD 1 , MITF, Mir20G, Mir204, Mir21 1 , Klf4, c- myc, KIf9, RARa, Pax2, Smad3, Sox4, Soxl l, Otxl, Pitx2, Nr2fl, Nr2f2, Tfec and any combination thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

Figs. 1A-B. Protocol to determine if iRPE conversion occurs through

transdifferentiation or spontaneous differentiation through PAX6 progenitors.

Figs. 2A-D. 13 DF and 8 DF RPE65⁺ mouse iRPE cells converted from MEFs with dox for 3 weeks followed by dox holiday for 2 weeks and iPSC-RPEs. (A) iRPE cells have a cobblestone morphology and dark brown appearance suggestive of pigment. (B) 13 DF and (C) 8 DF iRPEs from MEFs are X-gal positive. (D) Day 28 of mouse iPSCs differentiated into RPE-like cells.

Figs. 3A-C. . Mouse iRPE qPCR of (A) 13 DF, (B) 12 DF, and (C) 8 DF without any constitutive vectors reveal expression of mature RPE signature genes.

Figs. 4A-B. (A) HFLF cell clusters stained with DRAQ5 live nuclear stain at day 25. (B) qPCR of 13 DF human iRPE reveals expression of RPE signature.

Figs. 5A-I. . Micron HI integrated SD-OCT and fundus photography 3 months after MNU injury. (A-C) Top row of normal 4F2A retina demonstrates healthy appearance of retinal layers on OCT b-scan, and H&E section. Red line on brightfield fundus image correlates with b-scan image in middle column (inset shows optic nerve on OCT). (D-F) Middle row brightfield demonstrates increased visibility of subretinal layers due to photoreceptor atrophy consistent with other photoreceptor degeneration models. OCT and H&E demonstrate absence of ONL and PRS. (G-I) Lower row demonstrates reduced pigmentation on brightfield compared to MNU but more than normal. OCT and H&E demonstrates partial regeneration of outer retina with hyperrefiective band separating INL and ONL and blurring of the PRS. On H&E there are three blue nuclear layers (I) compared to two nuclear layers in (F).

Figs. A-B. (A) B wave amplitudes obtained longitudinally in scotopic conditions. 4F-induced recovery begins at 2 weeks and gradually increases over 2 months. (B)

Reconstituted a and b waves and oscillatory potentials in 4F-treated MNU injured mice at day 36 at 100 cd.s/m2.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is explained in greater detail below. This description is not intended to be a detailed catalog of all the different ways in which the invention may be implemented, or all the features that may be added to the instant invention. For example, features illustrated with respect to one embodiment may be incorporated into other embodiments, and features illustrated with respect to a particular embodiment may be deleted from that embodiment. In addition, numerous variations and additions to the various embodiments suggested herein will be apparent to those skilled in the art in light of the instant disclosure which do not depart from the instant invention. Hence, the following specification is intended to illustrate some particular embodiments of the invention, and not to exhaustively specify all permutations, combinations and variations thereof.

Definitions.

"Subjects" as used herein include any animal in which treatment of a disorder of the eye is necessary or desired. In some embodiments, a subject of this invention can be a mammalian subject, which can be a human subject. A subject of this invention can be male or female and may be of any race or ethnicity, including, but not limited to, Caucasian, African-American, African, Asian, Hispanic, Indian, etc. The subjects may be of any age, including newborn, neonate, infant, child, adolescent, adult, and geriatric. Subjects may also include animal subjects, particularly mammalian subjects such as canines, felines, bovines, caprines, equines, ovines, porcines, rodents (e.g. rats and mice), lagomorphs, primates (including non-human primates), etc., for veterinary medicine or pharmaceutical drug development purposes. The terms "therapeutically effective amount" and "effective amount" as used herein are synonymous unless otherwise indicated, and mean an amount of a protein, nucleic acid molecule, cell or composition of the present invention that is sufficient to improve the condition, disease, or disorder being treated and/or achieved the desired benefit or goal.

Determination of a therapeutically effective amount, as well as other factors related to effective administration of a protein, nucleic acid molecule or composition of the present invention to a subject of this invention, including dosage forms, routes of administration, and frequency of dosing, may depend upon the particulars of the condition that is encountered, including the subject and condition being treated or addressed, the severity of the condition in a particular subject, the particular compound being employed, the particular route of administration being employed, the frequency of dosing, and the particular formulation being employed. Determination of a therapeutically effective treatment regimen for a subject of this invention is within the level of ordinary skill in the medical or veterinarian arts. In clinical use, an effective amount may be the amount that is recommended by the U.S. Food and Drug Administration, or an equivalent foreign agency. The amount of active ingredient that can be combined with the carrier materials to produce a single dosage form varies depending upon the subject being treated and the particular mode of administration.

"Treat," "treating" or "treatment" as used herein refers to any type of action or administration that imparts a benefit to a subject that has a disease or disorder, including improvement in the condition of the patient (e.g., reduction or amelioration of one or more symptoms), delay in the progression of the disease, healing, reversal of the disease or disorder, etc.

"Pharmaceutically acceptable" as used herein means that the compound or composition is suitable for administration to a subject to achieve the treatments described herein, without unduly deleterious side effects in light of the severity of the disease and necessity of the treatment.

Administration of a protein, nucleic acid molecule, cell or composition of this invention can be by administration into the eye, for example by injection into the eye (i.e., intraocular injection, which can be for example, intraretinal injection, suprachoroidal injection, subretinal injection, intracomeal injection, intracameral injection and/or intravitreal injection). In some embodiments, administration may be by implant, via a matrix, via a gel, ointment, liquid drop or any combination thereof.

"Concurrently administering" or "concurrently administer" as used herein means that the two or more compounds or compositions are administered closely enough in time to produce a combined effect (that is, concurrently may be simultaneously, or it may be two or more events occurring within a short time period before or after each other, e.g., sequentially). Simultaneous concurrent administration may be carried out by mixing the compounds prior to administration, or by administering the compounds at the same point in time but at different anatomic sites and/or by using different routes of administration.

"Analog" as used herein means a protein that has the physiological activity of the parent protein thereof, and that includes one or more (e.g., two, three, four, five or six or more) amino acids different from the amino acid sequence of a naturally occurring parent protein. Such an analog preferably has at least about 70% of the physiological activity of the parent protein. Such different amino acids may be additions, substitutions, deletions, or combinations thereof, including addition of non-natural side-chain groups and backbone links. Modifications of proteins to produce analogs thereof are known. See, e.g., US Patent No. 7,323,543; see also US Patent Nos. 7,482,171; 7,459,152; and 7,393,919.

The terms "polypeptide," "peptide," and "protein," used interchangeably herein, refer to a polymeric form of amino acids of any length, which can include genetically coded and non-genetically coded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides having modified peptide backbones. The term includes fusion proteins, including, but not limited to, fusion proteins with a heterologous amino acid sequence, fusion proteins with heterologous and homologous leader sequences, with or without N-terminal methionine residues, immunologically tagged proteins, and the like, as are known in the art.

The terms "nucleic acid" and "polynucleotide" are used interchangeably herein and refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof. Non-limiting examples of polynucleotides include linear and circular nucleic acids, messenger RNA (mRNA), cDNA, recombinant polynucleotides, vectors, probes, and primers.

The term "operably linked" refers to functional linkage between molecules to provide a desired function. For example, "operably linked" in the context of nucleic acid molecules refers to a functional linkage between nucleic acid molecules to provide a desired function such as transcription, translation, and the like, e.g., a functional linkage between a nucleic acid expression control sequence (such as a promoter, signal sequence, or array of

transcription factor binding sites) and a polynucleotide sequence, wherein the expression control sequence affects transcription and/or translation of the polynucleotide sequence. As used herein the term "isolated" with reference to a cell, refers to a cell that is in an environment different from that in which the cell naturally occurs, e.g., where the cell naturally occurs in a multicellular organism, and the cell is removed from the multicellular organism, the cell is "isolated." An isolated genetically modified host cell can be present in a mixed population of genetically modified host cells, or in a mixed population comprising genetically modified host cells and host cells that are not genetically modified. For example, an isolated genetically modified host cell can be present in a mixed population of genetically modified host cells in vitro, or in a mixed in vitro population comprising genetically modified host cells and host cells that are not genetically modified.

A "host cell," as used herein, denotes an in vivo or in vitro cell (e.g., a eukaryotic cell cultured as a unicellular entity), which ceil can be, or has been, used as a recipient of a nucleic acid molecule (e.g., an exogenous nucleic acid molecule) or an exogenous polypeptide, and includes the progeny of the original cell which has been modified by introduction of the exogenous polypeptide or genetically modified by the nucleic acid molecule. It is understood that the progeny of a single cell may not necessarily be completely identical in morphology or in genomic or total DNA complement as the original parent cell, due to natural, accidental, or deliberate mutation.

The terms "genetic modification" and "genetically modified" refer to a permanent or transient genetic change induced in a cell following introduction of a nucleic acid molecule (i.e., a nucleic acid molecule exogenous to the cell). Genetic change ("modification") can be accomplished by incorporation of the nucleic acid molecule into the genome of the host cell, or by transient or stable maintenance of the nucleic acid molecule as an extrachromosomal element. Where the cell is a eukaryotic cell, a permanent genetic change can be achieved by introduction of the nucleic acid molecule into the genome of the cell. Suitable methods of genetic modification include viral infection, transfection, conjugation, protoplast fusion, electroporation, particle gun technology, calcium phosphate precipitation, direct

microinjection, and the like as are known in the art.

As used herein, the term "exogenous nucleic acid molecule" refers to a nucleic acid molecule that is not normally or naturally found in and/or produced by a cell in nature, and/or that is introduced into the cell (e.g., by electroporation, transfection, infection, lipofection, or any other means of introducing a nucleic acid molecule into a cell).

Before the present invention is further described, it is to be understood that this invention is not limited to particular embodiments described, as such may, of course, vary, It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, the preferred methods and materials are now described.

All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited.

As used herein, the terms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a cell" includes a single cell as well as a plurality of cells.

It is further noted that the claims may be drafted to exclude any element of this invention. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as "solely," "only" and the like in connection with the recitation of claim elements, or use of a negative limitation.

The present invention is based on the unexpected discovery that somatic cells can be directly induced with various transcription factors to transdifferentiate into retinal pigment epithelium cells, photoreceptor cells or retinal progenitor cells, respectively, both in vitro and in vivo. In particular, the present invention provides methods of inducing a somatic cell to transdifferentiate into a retinal pigment epithelium cell, a photoreceptor cell or a retinal progenitor cell in the absence of eggs, embryos, embryonic stem cells, induced pluripotent stem cells or embryonic germ cells.

Thus, in one aspect, the present invention provides a method of generating an induced retinal pigment epithelium cell, comprising introducing into a somatic cell one or more transcription factors selected from the group consisting of OTX2, FOXG1, Pax6, Smad6, Lhx2, HNF4aIpha, PKNOX2, Sox9, FoxDl, MITF, Mir200, Mir204, Mir211₅ Crx, Six3, Klf4, C-Myc, Klf9, RARa, Pax2, Smad3, Sox4, Soxl l, Otxl, Pitx2, Nr2fl, Nr2f2, Tfec and any combination thereof.

In a further aspect, the present invention provides a method of generating an induced photoreceptor cell, comprising introducing into a somatic cell one or more transcription factors selected from the group consisting of Ascll, Brn2_} Rax, Crx, Nrl, NeuroDl, Otx2, MhT24, FoxGl, Pax6, Ars2_s Sox2, Nr2e3, Ror-beta, Blimp 1, CBP, p300, TR 2, PIAS3, Nr2el, Nrldl, Vsx2, Six6, RBI, Mirl82, Mirl83, Pax7 and any combination thereof.

Also provided herein is a method of generating an induced retinal progenitor cell, comprising introducing into a somatic cell one or more transcription factors selected from the group consisting of Otx2, Rax, Ascll, Crx, Sox2, Vsx2 and any combination thereof.

In an additional aspect, the present invention provides a method of generating an induced retinal pigment epithelium cell, comprising introducing into a somatic cell the transcription factor OTX2 plus a cyclic AMP agonist molecule,

The methods described herein can further comprise introducing into the somatic cell the transcription factor HNF4a and/or further comprise introducing into the somatic cell one or more transcription factors selected from the group consisting of FOXG1, Smad6, Lhx2, H F4a and any combination thereof and/or further comprise introducing into the somatic cell one or more transcription factors selected from the group consisting of Klf4, c-myc, Klf9, RARa, Pax2, Smad3, Sox4, Soxl l, Otxl, Pitx2, Nr2fl, Nr2f2, Tfec and any combination thereof. In an additional aspect, the present invention provides a method of generating an induced photoreceptor cell, comprising introducing into a somatic cell one or more transcription factors selected from the group consisting of Ascll, Crx, Nrl, NeuroDl, Rax, Otx2, Mir 124, FoxGl, Pax6, Ars2 and any combination thereof. This method can further comprise introducing into the somatic cell one or more transcription factors selected from the group consisting of Nr2e3, Ror-beta, Blimpl, Mir200, CBP, p300, TRp2, PIAS3, Nr2el, Nrldl, Vsx2, Six6, RBI, Mir 182, Mir 183, Pax7 and any combination thereof.

In another aspect of this invention, a method is provided of generating an induced retinal progenitor cell, comprising introducing into a somatic cell the transcription factors mir200, mir2G4 and mir211. This method can further comprise introducing into the somatic cell the transcription factor MITF. These preceding methods can further comprise introducing into the somatic cell the transcription factor Smad6. These preceding methods can further comprise introducing into the somatic cell the transcription factors HNF4 alpha, Sox9, P NOX2, Pax6, FoxDl, FoxGl and Otx2. These preceding methods can further comprise introducing into the somatic cell the transcription factor Lhx2. These preceding methods can further comprise introducing into the somatic cell a transcription factor selected from the group consisting of Sox2, Ikzfl, Ascll and any combination thereof.

The present invention additionally provides an induced retinal pigment epithelium cell, an induced photoreceptor cell and/or an induced retinal progenitor cell produced by the respective methods of this invention. Also provided herein is a population of induced retinal pigment epithelium cells, a population of induced photoreceptor cells and a population of induced retinal progenitor cells produced by the respective methods of this invention.

Additional aspects of this invention provide method claims, including, in one embodiment, a method of treating a disorder of the eye in a subject (e.g., a subject in need thereof), comprising delivering to the eye(s) of the subject an effective amount of a cell of this invention.

A cell of this invention can be a somatic cell. In some embodiments, the cell can be a fibroblast. In some embodiments, the cell can be a white blood cell. The cell can be a neonatal cell, germ cell, an adult cell, a post-natal cell, a non-retinal cell, a retinal cell, a pluripotent cell, and any combination thereof.

In the methods of this invention, introducing one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, etc.) transcription factor of this invention can comprise, consist essentially of or consist of genetically modifying the somatic cell either in vitro, in vivo or both with one or more nucleic acid molecules comprising a nucleotide sequence encoding said transcription factor(s) Any number of nucleic acid molecules can be introduced into the cell to achieve the desired result of having the one or more transcription factors produced in the cell via expression of the one or more nucleic acid molecules.

In the methods of this invention, the induced cells can be analyzed for characteristics of endogenous retinal pigment epithelium cells, endogenous photoreceptor cells, or endogenous retinal progenitor cells, respectively. Noniimiting examples of characteristics of an endogenous retinal pigment epithelium cell include gene and protein expression of RPE65, Cralbp, Bestrophin, tyrosinase, resting membrane and transepithelial potential, polarized secretion of VEGF and PEDF, and phagocytosis. Noniimiting examples of characteristics of an endogenous photoreceptor cell include gene and protein expression of rhodopsin, recoverin, peripherin, converting light stimulus into an electrical impulse Noniimiting examples of characteristics of an endogenous retinal progenitor cell include the ability to differentiate into retinal neuronal subtypes, gene and protein expression of Nestin, Sox2, ChxlO, Pax6, Six6, Six3, or Rax.

The methods of this invention can be carried out under conditions as are known in the art to produce induced retinal pigment epithelium cells, induced photoreceptor cells or included retinal progenitor cells that exhibit one or more of the characteristics of the respective endogenous cell. Assays to establish the presence of such characteristics are as described herein and as are known in the art.

In some embodiments, a transcription factor can be introduced into a cell of this invention as an exogenous protein and in some embodiments, the exogenous protein can comprise a heterologous protein transduction domain, which can be linked to the exogenous protein covalently or non-covalently. A protein transduction domain or PTD refers to a polypeptide, polynucleotide, carbohydrate, and/or organic or inorganic compound that facilitates traversing a lipid bilayer, micelle, cell membrane, organelle membrane, or vesicle membrane. A PTD attached to another molecule facilitates the molecule traversing a membrane, for example going from extracellular space to intracellular space, or from cytosol to within an organelle. In some embodiments, a PTD can be covalently linked to the amino terminus of a transcription factor of this invention. In some embodiments, a PTD can be covalently linked to the carboxyl terminus of a transcription factor of this invention.

Exemplary protein transduction domains include but are not limited to a minimal undecapeptide protein transduction domain (corresponding to residues 47-57 of HIV- 1 TAT comprising YGRKKRRQRRR); a polyarginine sequence comprising a number of arginines sufficient to direct entry into a cell (e.g., 3, 4, 5, 6, 7, 8, 9, 10, or 10-50 arginines); a VP22 domain (Zender et al. Cancer Gene Ther 9(6):489-96 (2002)); a Drosophilo antennapedia protein transduction domain (Noguchi et al. Diabetes 52(7): 1732-1737 (2003)); a truncated human calcitonin peptide (Trehin et al. Pharm. Research 21 : 1248-1256 (2004)); polylysine (Wender et al. PNAS 97:13003-13008 (2000)); RRQRRTS LM R Transportan

GWTLNSAGYLLGKINL ALAALAKKIL;

KAL A WE AKLAKALA AL AKHL AKAL AK ALKCE A ; and RQIKIWFQNRRMKWKK. Exemplary PTDs include but are not limited to, YGRKKRRQRRR, RKKRRQRRR, an arginine homopolymer of from 3 arginine residues to 50 arginine residues,

YARAAARQARA; THRLPRRRRRR; and GGRRARRRRRR.

In some embodiments, a transcription factor of this invention can comprise an arginine homopolymer of from 3 arginine residues to 50 arginine residues, e.g., from 3 to 6 arginine residues, from 6 to 10 arginine residues, from 10 to 20 arginine residues, from 20 to 30 arginine residues, from 30 to 40 arginine residues, or from 40 to 50 arginine residues. In some embodiments, a transcription factor of this invention can comprise six Arg residues covalently linked (e.g., by a peptide bond) at the amino terminus of the reprogramming factor polypeptide. In some embodiments, a transcription factor of this invention can comprise six Arg residues covalently linked (e.g., by a peptide bond) at the carboxyl terminus of the transcription factor.

Exogenous transcription factors that are introduced into a somatic cell of this invention can be purified, e.g., at least about 75% pure, at least about 80% pure, at least about 85% pure, at least about 90% pure, at least about 95% pure, at least about 98% pure, at least about 99% pure, or more than 99% pure, e.g., free of proteins other than transcription factor(s) being introduced into the cell and free of macromolecules other than the

transcription factor(s) being introduced into the cell.

In some embodiments, introduction of one or more transcription factor of this invention into a somatic cell is achieved by genetic modification of the somatic cell with one or more exogenous nucleic acids comprising one or more nucleotide sequences encoding one or more transcription factors of this invention.

The one or more exogenous nucleic acids comprising nucleotide sequences encoding the one or more transcription factors of this invention can be used in the methods of this invention in the form a recombinant expression vector, where suitable vectors include, without limitation, e.g., recombinant retroviruses, lentiviruses, alphaviruses, adeno-associated viruses and adenoviruses; retroviral expression vectors, lentiviral expression vectors, alphavirus expression vectors, adeno-associated virus expression vectors, nucleic acid expression vectors, and plasmid expression vectors. In some embodiments, the one or more exogenous nucleic acid molecules can be integrated into the genome of a somatic cell and its progeny. In some embodiments, the one or more exogenous nucleic acid molecules can be present in an episomal state in the somatic cell and its progeny. In some embodiments, an endogenous, natural version of the transcription factor-encoding nucleic acid may already exist in the somatic cell but an additional "exogenous nucleic acid molecule" is added to the somatic cell to increase expression of the transcription factor. In other embodiments, the transcription factor-encoding nucleic acid molecule encodes a transcription factor polypeptide having an amino acid sequence that differs by one or more amino acids from a polypeptide encoded by an endogenous transcription factor-encoding nucleic acid within the somatic cell.

In some embodiments, a somatic cell of this invention is genetically modified with two or more (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, etc.) separate expression constructs (expression vectors), each comprising a nucleotide sequence encoding a transcription factor of this invention. In some embodiments, an expression construct can comprise nucleotide sequences encoding two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, etc.) of the transcription factors of this invention.

In some embodiments, one or more exogenous nucleic acids comprising nucleotide sequences encoding one or more of the transcription factors of this invention is introduced into a single somatic cell (e.g., a single somatic host cell) in vitro. In some embodiments, one or more exogenous nucleic acids comprising nucleotide sequences encoding the transcription factors of this invention is introduced, into a population of somatic cells (e.g., a population of host somatic cells) in vitro. In some embodiments, one or more exogenous nucleic acids comprising nucleotide sequences encoding one or more of the transcription factors of this invention can be introduced into a somatic cell (e.g., a single somatic cell or a population of somatic cells) in vivo.

Where a population of somatic cells of this invention is genetically modified (in vitro, ex vivo or in vivo) with one or more exogenous nucleic acids comprising nucleotide sequences encoding one or more transcription factor of this invention, the one or more exogenous nucleic acids can be introduced into greater than 20% of the total population of somatic cells, e.g., 25%, 30%, 35%, 40%, 44%, 50%, 57%, 62%, 70%, 74%, 75%, 80%, 90%, or other percent of cells greater than 20%.

In some embodiments, the one or more nucleic acid molecules comprising nucleotide sequences encoding one or more transcription factors of this invention can be present as an expression construct that provides for production of the one or more transcription factor polypeptides in the genetically modified host somatic cell. In some embodiments, the expression construct can be a viral construct, e.g., a recombinant adeno-associated virus construct (see, e.g., U.S. Pat No. 7,078,387), a recombinant adenoviral construct, a recombinant lentiviral construct, etc., as are well known in the art.

Nonlimiting examples of suitable expression vectors include viral vectors (e.g., viral vectors based on vaccinia virus; poliovirus; adenovirus (see, e.g., Li et al. Invest Ophthalmol Vis Sci 35:2543 2549 (1994); Borras et al. Gene Ther 6:515 524 (1999) Li and Davidson PNAS 92:7700 7704 (1995); Sakamoto et al. HGene Ther 5:1088-1097 (1999); PCT

Publication Nos. WO 94/12649, WO 93/03769; WO 93/19191; WO 94/28938; WO 95/11984 and WO 95/00655); adeno-associated virus (see, e.g., Ali et al., Hum Gene Ther 9:8186, 1998, Flarmery et al., PNAS 94:6916 6921, 1997; Bennett et al., Invest Opthalmol Vis Sci 38:2857 2863, 1997; Jomary et al., Gene Ther 4:683 690, 1997, Rolling et al., Hum Gene Ther 10:641 648, 1999; Ali et ah, Hum Mol Genet 5:591 594, 1996; Srivastava in WO 93/09239, Samulski et al., J Virol. (1989) 63:3822-3828; Mendelson et al., Virology (1988) 166:154-165; and Flotte et ai, PNAS (1993) 90:10613-10617); SV40; herpes simplex virus; human immunodeficiency virus (see, e.g., Miyoshi et al., PNAS 94:10319 23, 1997;

Takahashi et al., J Virol 73:7812 7816, 1999); a retroviral vector (e.g., Murine Leukemia Virus, spleen necrosis virus, and vectors derived from retroviruses such as Rous Sarcoma Virus, Harvey Sarcoma Virus, avian leukosis virus, a lentivirus, human immunodeficiency virus, myeloproliferative sarcoma virus, and mammary tumor virus); and the like.

Numerous suitable expression vectors are known to those of skill in the art, and many are commercially available. The following vectors are provided by way of example; for eukaryotic host cells: XTl, pSG5 (Stratagene), pSVK3, pBPV, pMSG, and pSVLSV40 (Pharmacia). However, any other vector may be used so long as it is compatible with the host cell.

Depending on the host/vector system utilized, any of a number of suitable

transcription and translation control elements, including constitutive and inducible promoters, transcription enhancer elements, transcription terminators, etc., may be used in an expression vector of this invention (see e.g., Bitter et al. (1987) Methods in Enzymology 153:516-544).

In some embodiments, a transcription factor-encoding nucleotide sequence of this invention can be operably linked to a control element, e.g., a transcriptional control element, such as a promoter. The transcriptional control element is functional in a eukaryotic cell, e.g., a mammalian cell. Suitable transcriptional control elements include promoters and enhancers. In some embodiments, the promoter is constitutively active. In other embodiments, the promoter is inducible.

Non-limiting examples of suitable eukaryotic promoters (promoters functional in a eukaryotic cell) include CMV immediate early, HSV thymidine kinase, early and late SV40, long terminal repeats (LTRs) from retrovirus, and mouse metallothionein-I.

In some embodiments, a transcription factor-encoding nucleotide sequence of this invention can be operably linked to a transcriptional regulator element (TRE), where TREs include promoters and enhancers.

Selection of the appropriate vector and promoter is well within the level of ordinary skill in the art. The expression vector may also contain a ribosome binding site for translation initiation and a transcription terminator. The expression vector may also include appropriate sequences for amplifying expression.

Examples of suitable mammalian expression vectors (expression vectors suitable for use in mammalian host cells) include, but are not limited to: recombinant viruses, nucleic acid vectors, such as plasmids, bacterial artificial chromosomes, yeast artificial

chromosomes, human artificial chromosomes, cDNA, cRNA, and polymerase chain reaction (PCR) product expression cassettes. Examples of suitable promoters for driving expression of a transcription factor-encoding nucleotide sequence of this invention include, but are not limited to, retroviral long terminal repeat (LTR) elements; constitutive promoters such as CMV, HSV1-TK, SV40, EF-la, β-actin; phosphoglycerol kinase (PGK), and inducible promoters, such as those containing Tet-operator elements. In some cases, the mammalian expression vector(s) encodes, in addition to exogenous transcription factor polypeptides, a marker gene that facilitates identification or selection of cells that have been transfected or infected. Examples of marker genes include, but are not limited to, genes encoding fluorescent proteins, e.g., enhanced green fluorescent protein, Ds-Red (DsRed: Discosoma sp. red fluorescent protein (RFP) (Bevis and GlickMtf. Biotechnol. 20:83 (2002)), yellow fluorescent protein, and cyanofluorescent protein; and genes encoding proteins conferring resistance to a selection agent, e.g., a neomycin resistance gene, a puromycin resistance gene, a blasticidin resistance gene, and the like.

Examples of suitable viral vectors include, but are not limited, viral vectors based on retroviruses (including lentiviruses), alphavir ses, adenoviruses; and adeno-associated viruses. An example of a suitable retrovirus -based vector is a vector based on murine moloney leukemia virus (MMLV); however, other recombinant retroviruses may also be used, e.g., Avian Leukosis Virus, Bovine Leukemia Virus, Murine Leukemia Virus (MLV), Mink-Cell focus-Inducing Virus, Murine Sarcoma Virus, Reticuloendotheiiosis virus, Gibbon Abe Leukemia Virus, Mason Pfizer Monkey Virus, or Rous Sarcoma Virus, see, e.g., U.S. Pat No. 6,333,195.

In other cases, the retrovirus-based vector is a lenti virus-based vector, (e.g., Human Immunodeficiency Virus-1 (HIV-1); Simian Immunodeficiency Virus (SIV); or Feline Immunodeficiency Virus (FIV)), see, e.g., Johnston et al. Journal of Virology 73(6):4991- 5000 (1999) (FIV); Negre et al. (2002) Current Topics in Microbiology and Immunology 261:53-74 (2002) (SIV); Naldini et al., Science 272:263-267 (1996) (HIV).

The recombinant retrovirus may comprise a viral polypeptide (e.g., retroviral env) to aid entry into the target cell. Such viral polypeptides are well-established in the art, see, e.g., U.S. Pat. No. 5,449,614. The viral polypeptide may be an amphotropic viral polypeptide, e.g., amphotropic env, which aids entry into cells derived from multiple species, including cells outside of the original host species. The viral polypeptide may be a xenotropic viral polypeptide that aids entry into cells outside of the original host species. In some

embodiments, the viral polypeptide is an ecotropic viral polypeptide, e.g., ecotropic env, which aids entry into cells of the original host species.

Examples of viral polypeptides capable of aiding entry of retroviruses into cells include but are not limited to MMLV amphotropic env, MMLV ecotropic env, MMLV xenotropic env, vesicular stomatitis virus-G protein (VSV-G), HIV-1 env, Gibbon Ape Leukemia Virus (GALV) env, RD 114, FeLV-C, FeLV-B, MLV 10A1 env gene, and variants thereof, including chimeras (see e.g., Yee et al. (1994) Methods Cell Biol. Pt A:99-l 12 (1994) (VSV-G); U.S. Pat. No. 5,449,614). In some cases, the viral polypeptide is genetically modified to promote expression or enhanced binding to a receptor.

Methods of producing recombinant viruses and their uses are well established; see, e.g., U.S. Pat. Nos. 5,834,256; 6,910,434; 5,591,624; 5,817,491; 7,070,994; and 6,995,009. Many methods begin with the introduction of a viral construct into a packaging cell line. The viral construct may be introduced into a somatic cell of this invention by any method known in the art, including but not limited to: a calcium phosphate method, a lipofection method (Feigner et al. Proc. Natl. Acad. Sci. U.S.A. 84:7413-7417(1987), an electroporation method, microinjection, Fugene transfection, and the like, as well as any method described herein.

A nucleic acid construct can be introduced into a somatic cell of this invention using a variety of well known techniques, such as non- viral based transfection of the cell. In an exemplary aspect the construct is incorporated into a vector and introduced into a host cell. Introduction into the cell may be performed by any non-viral based transfection known in the art, such as, but not limited to, electroporation, calcium phosphate mediated transfer, nucleofection, sonoporation, heat shock, magnetofection, liposome mediated transfer, microinjection, microprojectile mediated transfer (nanoparticles), cationic polymer mediated transfer (DEAE-dextran, polyethylenimine, polyethylene glycol (PEG) and the like) or cell fusion. Other methods of transfection include transfection reagents such as Lipofectamine™, Dojindo Hilymax™, Fugene™, jetPEI™, Effectene™, and DrearnFect™.

Suitable amino acid sequences of the transcription factors of this invention include amino acid sequences having at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100%, amino acid sequence identity to an amino acid sequence set forth in Tables 1 and 2.

Suitable nucleotide sequences encoding a transcription factor of this invention include nucleotide sequences having at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100%, nucleotide sequence identity to a nucleotide sequence set forth in Tables 1 and 2.

The present invention provides genetically modified somatic cells, including isolated genetically modified somatic cells, wherein a genetically modified somatic cell of this invention comprises, consists essentially of or consists of (i.e., has been genetically modified with) one or more exogenous nucleic acids comprising one or more nucleotide sequences encoding one or more transcription factors of this invention. In some embodiments, a genetically modified somatic cell is in vitro. In some embodiments, a genetically modified somatic cell is a human cell or is derived from a human cell. In some embodiments, a genetically modified somatic cell is a rodent cell or is derived from a rodent cell. The present invention further provides progeny of a genetically modified somatic cell of this invention, wherein the progeny can comprise the same exogenous nucleic acid as the genetically modified somatic cell from which it was derived. The present invention further provides a composition comprising a genetically modified cell of this invention.

The present invention further provides induced retinal pigment epithelium cells (RPEs), induced photoreceptor (PR) cells and induced retinal progenitor cells derived from a genetically modified somatic cell of this invention. Because an induced RPE, induced PR cell and induced retinal progenitor cell is derived from a genetically modified somatic cell, an induced RPE, induced PR cell and induced retinal progenitor cell is also genetically modified. Thus, the present invention provides a genetically modified induced RPE, induced PR cell and induced retinal progenitor cell, respectively, that comprises one or more exogenous nucleic acids comprising one or more nucleotide sequences encoding one or more transcription factors of this invention. In some embodiments, a genetically modified induced RPE, induced PR cell and induced retinal progenitor cell, respectively, is in vitro. In some embodiments, a genetically modified induced RPE, induced PR cell and induced retinal progenitor cell, respectively, is a human cell or is derived from a human cell. In some embodiments, a genetically modified induced RPE, induced PR cell and induced retinal progenitor cell, respectively, is a rodent cell or is derived from a rodent cell. The present disclosure further provides progeny of a genetically modified induced RPE, induced PR cell and induced retinal progenitor cell, respectively, wherein the progeny can comprise the same exogenous nucleic acid as the genetically modified induced RPE,. induced PR cell and induced retinal progenitor cell, respectively, from which it was derived. The present invention further provides a composition comprising a genetically modified induced RPE, induced PR cell and/or induced retinal progenitor cell, respectively, of this invention.

The present invention provides a composition comprising a genetically modified somatic cell (e.g., a genetically modified somatic cell; progeny of a genetically modified somatic cell; an induced RPE, induced PR cell and induced retinal progenitor cell; progeny of an induced RPE, induced PR cell and induced retinal progenitor cell) and a suitable carrier, A composition of this invention can comprise a genetically modified cell of this invention and can in some embodiments comprise one or more additional components, which components are selected based in part on the intended use of the genetically modified cell of this invention. Suitable components include, but are not limited to, salts; buffers; stabilizers; protease-inhibiting agents; cell membrane- and/or cell wall-preserving compounds, e.g., glycerol, dimethylsulfoxide, etc.; nutritional media appropriate to the cell; and the like.

In some embodiments, a composition of this invention can comprise a genetically modified cell of this invention and a matrix (a "genetically modified cell/matrix

composition"), where a genetically modified cell of this invention is associated with the matrix. The term "matrix" refers to any suitable carrier material to which the genetically modified cells are able to attach themselves or adhere in order to form a cell composite. In some embodiments, the matrix or carrier material is present already in a three-dimensional form desired for later application.

For example, a matrix (also referred to as a "biocompatible substrate") can be a material that is suitable for implantation into a subject. A biocompatible substrate does not cause toxic or injurious effects once implanted in the subject. In one embodiment, the biocompatible substrate is a polymer with a surface that can be shaped into a desired structure or part of a desired structure. The biocompatible substrate can provide a supportive framework that allows cells to attach to it and/or grow on it.

Suitable matrix components include, e.g., collagen; gelatin; fibrin; fibrinogen;

laminin; a glycosaminoglycan; elastin; hyaluronic acid; a proteoglycan; a glycan; poly(lactic acid); poly (vinyl alcohol); poly(vinyl pyrrolidone); poly(ethylene oxide); cellulose; a cellulose derivative; starch; a starch derivative; poly(caprolactone); poly(hydroxy butyric acid); mucin; and the like. In some embodiments, the matrix comprises one or more of collagen, gelatin, fibrin, fibrinogen, laminin, and elastin; and can further comprise a non- proteinaceous polymer, e.g., can further comprise one or more of poly(lactic acid), poly( vinyl alcohol), poly(vinyl pyrrolidone), poly(ethylene oxide), poly(caprolactone), poly(hydroxy butyric acid), cellulose, a cellulose derivative, starch, and a starch derivative. In some embodiments, the matrix comprises one or more of collagen, gelatin, fibrin, fibrinogen, laminin, and elastin; and can further comprise hyaluronic acid, a proteoglycan, a

glycosaminoglycan, or a glycan. Where the matrix comprises collagen, the collagen can comprise type I collagen, type II collagen, type III collagen, type V collagen, type XI collagen, and combinations thereof.

In some embodiments, the matrix can be a hydrogei. A suitable hydrogei is a polymer of two or more monomers, e.g., a homopolymer or a heteropolymer comprising multiple monomers. Non-limiting examples of suitable hydrogei monomers include the following: lactic acid, glycolic acid, acrylic acid, 1-hydroxyethyl methacrylate (HEMA), ethyl methacrylate (E A), propylene glycol methacrylate (PEMA), acrylamide (AAM), N- vinylpyrrolidone, methyl methacrylate (MMA), glycidyl methacrylate (GDMA), glycol methacrylate (GMA), ethylene glycol, fumaric acid, and the like. Common cross linking agents include tetraethylene glycol dimethacrylate (TEGD A) and Ν,Ν'- methylenebis acrylamide. The hydrogei can be homopolymeric, or can comprise co-polymers of two or more of the aforementioned polymers. Exemplary hydrogels include, but are not limited to, a copolymer of poly(ethylene oxide) (PEO) and poly(propylene oxide) (PPO); Pluronic™ F-127 (a difunctional block copolymer of PEO and PPO of the nominal formula ΕΟιοο-Ρθ65-ΕΟιοο, where EO is ethylene oxide and PO is propylene oxide); poloxamer 407 (a tri-block copolymer consisting of a central block of poly(propylene glycol) flanked by two hydrophilic blocks of poly(ethylene glycol)); a poly(ethylene oxide)-poly(propylene oxide)- poly(ethylene oxide) co-polymer with a nominal molecular weight of 12,500 Daltons and a PEOrPPO ratio of 2:1); a poly(N~isopropyiacrylamide)-base hydrogei (a PNIPAAm-based hydrogel); a PNiPAAm-acrylic acid co-polymer (PNIPAAm-co-AAc); poly(2-hydroxyethyl methacrylate); polyvinyl pyrrolidone); and the like,

A genetically modified cell/matrix composition of this invention can further comprise one or more additional components, wherein suitable additional components include, e.g., a growth factor; an antioxidant; a nutritional transporter (e.g., transferrin); a polyamine (e.g., glutathione, spermidine, etc.); and the like.

The cell density in a genetically modified cell/matrix composition of this invention can range from about 10² cells/mm³ to about 10⁹ cells/mm³, e.g., from about 10^a cells/mm³ to about 10⁴ cells/mm³, from about 10⁴ cells/mm³ to about 10⁶ cells/mm³, from about 10⁶ cells/mm³ to about 10⁷ cells/mm³, from about 10⁷ cells/mm³ to about 10^s cells/mm³, or from about 10⁸ cells/mm³ to about 10⁹ cells/mm³.

The matrix can take any of a variety of forms, or can be relatively amorphous. For example, the matrix can be in the form of a sheet, a cylinder, a sphere, etc., as are known in the art.

The present disclosure provides an implantable device that comprises a genetically modified cell of this invention, a composition of this invention, one or more transcription factors of this invention and/or one or more nucleic acid molecules encoding one or more transcription factors of this invention. The cell composition, transcription factor and/or nucleic acid molecules encoding one or more transcription factors can be coated onto a surface of the implantable device, or can be contained within a reservoir in the implantable device and in some embodiments, the reservoir can be designed to allow for elution of the cells, compositions, transcription factors and/or nucleic acid molecules from the reservoir.

When the implantable device is at a site in a subject, nucleic acid molecules and/or transcription factors present in the implantable device can leave the implantable device, and the transcription factors and/or nucleic acid molecules enter into a cell of the subject at or near the site of the implantable device. Thus, an implantable device of this invention, when implanted in a subject of this invention, can provide for introduction of transcription factors and/or nucleic acid molecules encoding same, into a cell of the subject at or near the site of implant, and can thereby provide for inducing the cell of the subject to become a RPE, a PR cell or a retinal progenitor cell, e.g., in the eye of a subject.

A composition of this invention can include a pharmaceutically acceptable carrier. Suitable carriers include, for example, water, saline, dextrose, glycerol, ethanol, or the like, and combinations thereof. In addition, if desired, the carrier can contain minor amounts of auxiliary substances such as wetting or emulsifying agents or pH buffering agents. Actual methods of preparing such compositions and/or formulations are known, or will be apparent, to those skilled in the art. See, e.g., Remington's Pharmaceutical Sciences. Mack Publishing Company, Easton, Pa., latest edition.

Pharmaceutically acceptable excipients, such as vehicles, adjuvants, carriers or diluents, are well known in the art. Moreover, pharmaceutically acceptable auxiliary substances, such as pH adjusting and buffering agents, tonicity adjusting agents, stabilizers, wetting agents and the like, are well known in the art.

In some embodiments, a composition of this invention can be formulated as a controlled release formulation. Sustained-release preparations may be prepared using methods well known in the art. Non-limiting examples of sustained-release matrices include polyesters, copolymers of L-glutamic acid and ethyl -L-glutamate, non-degradable ethylene- vinyl acetate, hydrogels, polylactides, degradable lactic acid-glycolic acid copolymers and poly-D-(-)-3-hydroxybutyric acid. Possible loss of biological activity and possible changes in activity of a polypeptide or a nucleic acid comprised in sustained-release preparations may be prevented by using appropriate additives, by controlling moisture content and/or by developing specific polymer matrix compositions.

A composition of this invention can further comprise one or more therapeutic agents. Nonlimiting examples of therapeutic agents of this invention include anti-inflammatory agents, immunosuppressive agents and any combination thereof.

A genetically modified somatic cell of this invention can be used to treat a subject in need of such treatment. Similarly, an induced RPE, induced PR cell and or induced retinal progenitor cell of this invention can be used to treat a subject in need of such treatment. A cell of this invention can be introduced into a recipient subject (e.g., a subject in need of treatment), where introduction of the cell(s) into the subject treats a condition or disorder in the subject. Thus, in some embodiments, a method of treatment involves administering to a subject in need thereof a population of genetically modified somatic cells of this invention. In some embodiments, a method of treatment of this invention involves administering to a subject in need thereof a population of induced RPEs, induced PR cells and/or induced retinal progenitor cells of this invention. The cells can be from the subject or the cells can be from an individual other than the subject.

In some embodiments, the present disclosure provides a method for performing cell transplantation in a recipient subject in need thereof, the method generally involving; (i) generating an induced RPE, an induced PR cell and/or an induced retinal progenitor cell from a somatic cell obtained from a donor, wherein the donor is immunocompatible with the recipient subject; and (ii) transplanting one or more of the induced cells of this invention into the recipient subject. In some embodiments, the recipient subject and the donor are the same individual. In some embodiments, the recipient subject and the donor are not the same individuals.

In some embodiments, the present disclosure provides a method for performing cell transplantation in a recipient subject in need thereof, comprising; (i) genetically modifying a somatic cell with one or more nucleic acids comprising nucleotide sequences encoding one or more transcription factors of this invention, wherein the somatic cells are obtained from a donor, wherein the donor is immunocompatible with the recipient subject; and (ii) transplanting one or more of the genetically modified somatic cells into the recipient subject. In some embodiments, the recipient subject and the donor are the same individual. In some embodiments, the recipient subject and the donor are not the same individuals.

In some embodiments, the present invention provides a method for performing cell transplantation in a subject in need thereof, comprising: (a) modifying a somatic cell by introducing into the somatic cell one or more transcription factors and/or one or more nucleic acid molecules encoding one or more transcription factors of this invention; and (a) transplanting one or more of the modified somatic cells into the subject.

The present disclosure provides methods of treating a disorder of the eye in an individual, comprising administering to a subject in need thereof a therapeutically effective amount of: a) a population of induced RPEs, a population of induced PR cells and/or a population of induced retinal progenitor cells prepared according to the methods of this invention; and/or b) a population of genetically modified somatic cells prepared according to the methods of this invention.

Non-limiting examples of a disorder of the eye that can be treated according to the methods of this invention include age-related macular degeneration, inherited macular degeneration, cystoid macular edema, retinal detachment, vascular occlusion, photoreceptor cell degeneration, infection, vision loss and any combination thereof.

For administration to a mammalian subject, a population of induced RPEs induced PR cells, induced retinal progenitor cells and/or a population of genetically modified somatic cells, generated using methods of the present invention can be formulated as a pharmaceutical composition. A pharmaceutical composition can be a sterile aqueous or non-aqueous solution, suspension or emulsion, which additionally comprises a physiologically acceptable carrier (i.e., a non-toxic material that does not interfere with the activity of the cells). Any suitable carrier known to those of ordinary skill in the art may be employed in a pharmaceutical composition of this invention. The selection of a carrier will depend, in part, on the nature of the substance (i.e., cells or chemical compounds) being administered.

Representative carriers include physiological saline solutions, gelatin, water, alcohols, natural or synthetic oils, saccharide solutions, glycols, injectable organic esters such as ethyl oleate or a combination of such materials. Optionally, a pharmaceutical composition may additionally contain preservatives and/or other additives such as, for example, antimicrobial agents, anti-oxidants, chelating agents and/or inert gases, and/or other active ingredients.

A unit dosage form of an induced RPE population, an induced PR cell population or a retinal progenitor cell population of this invention can contain from about 10,000 to about 10,000,000 cells. Dosage ranges for a nucleic acid molecule of this invention and a protein of this invention can be readily determined by one of ordinary skill in the art, based on information available regarding administration of nucleic acid molecules to the eye and administration of proteins to the eye. As one nonlimiting example, a dosage range of a nucleic acid molecule administered via an AAV vector can be from about 1.5 10⁹ to about 1.5xl0^!2. (See, e.g., Maguire et al. "Age dependent effects of RPE65 gene therapy for

Leber's congenital amaurosis: a phase 1 dose-escalation trial" Lancet 374:1597-1605 (2009)).

An induced cell population of this invention and/or a population of genetically modified somatic cells of this invention can be cryopreserved according to routine

procedures. For example, cryopreservation can be carried out on from about one to ten million cells in "freeze" medium which can include a suitable proliferation medium, 10% BSA and 7.5% dimethylsulfoxide. Cells are centrifuged. Growth medium is aspirated and replaced with freeze medium. Cells are resuspended as spheres. Cells are slowly frozen, by, e.g., placing in a container at -80°C. Cells are thawed by swirling in a 37°C bath,

resuspended in fresh proliferation medium, and grown as described herein.

Also provided herein is a method of reprogramming a cell in the eye of a subject into an induced retinal pigment epithelium cell, comprising introducing into the cell of the eye one or more transcription factors selected from the group consisting of OTX2, FOXGl, Pax6, Smad6, Lhx2, HNF4a, PKNOX2, Sox9, FoxDl, MITF, Mir200, Mir204, Mir211, Klf4, c- myc, Klf9, RARa, Pax2, Smad3, Sox4, Soxl 1 , Otxl, Pitx2, Nr2fl, Nr2f2, Tfec and any combination thereof.

Further provided herein is a method of reprogramming a cell in the eye of a subject into an induced photoreceptor cell, comprising introducing into the cell of the eye one or more transcription factors selected from the group consisting of Ascl , Crx, Nrl, NeuroDl, Rax, Otx2, Mirl24, FoxGl, Pax6, Ars2, Nr2e3, Ror-beta, Blimpl, Mir200, CBP, p300, TRp2, PIAS3, Nr2el, Nrldl, Vsx2, Six6, RB , Mirl82, Mirl83, Pax7 and any combination thereof.

Another aspect of this invention provides a method of reprogramming a cell in the eye of a subject into an induced retinal progenitor cell, comprising introducing into the cell of the eye one or more transcription factors selected from the group consisting of Otx2, Rax, Ascl 1 , Crx, Sox2, Vsx2 and any combination thereof.

Additionally provided herein is a method of reprogramming a cell in the eye of a subject into an induced retinal pigment epithelium cell, comprising introducing into the cell of the eye the transcription factors OTX2, Sox9, FoxDl and MITF. This method can further comprise the steps of introducing into the cell the transcription factor PKNOX2. The preceding methods can further comprise introducing into the cell the transcription factors Mir200, Mir204 and Mir211. The preceding methods can further comprise introducing into the cell one or more transcription factors selected from the group consisting of FOXG1, Smad6, Lhx2, HNF4a and any combination thereof. The preceding methods can further comprising introducing into the cell one or more transcription factors selected from the group consisting of Klf4, c-myc, Klf , RARa, Pax2, Smad3, Sox4, Soxl l, Otxl, Pitx2, Nr2fl, Nr2f2_f Tfec and any combination thereof.

Further aspects of this invention include a method of reprogramming a cell in the eye of a subject into an induced photoreceptor cell, comprising introducing into the cell of the eye one or more transcription factors selected from the group consisting of Ascl 1 , Crx, Nrl_}

NeuroDl, Rax, Otx2, Mir 124, FoxGl, Pax6, Ars2 and any combination thereof. This method can further comprise introducing into the cell one or more transcription factors selected from the group consisting of Nr2e3, Ror-beta, Blimpl, Mir200, CBP, p300, TRp2, PI AS 3, Nr2el, Nrldl, Vsx2, Six6, RBI, Mirl82, Mirl83, Pax7 and any combination thereof.

Additionally provided herein is a method of reprogramming a cell in the eye of a subject into an induced retinal progenitor cell, comprising introducing into the cell of the eye the transcription factors mir200, mir204 and mir211. This method can further comprise introducing into the cell the transcription factor MITF. The preceding methods can further comprise introducing into the cell the transcription factor Smad6. These preceding methods can further comprise introducmg into the cell the transcription factors HNF4alpha, Sox9, PKNOX2, Pax6, FoxDl, FoxGl and Otx2. These preceding methods can further comprise introducing into the cell the transcription factor Lhx2. These preceding methods can further comprise introducing into the cell a transcription factor selected from the group consisting of Sox2, Ikzfl, Ascll and any combination thereof. A method of reprogramming a preexisting retinal pigment epithelium (RPE) cell having no function or reduced/decreased/diminished function (e.g., an RPE cells that is damaged, diseased and/or aged) into a RPE cell having normal function or

increased/improved/enhanced function (e.g., compared to a damaged, diseased and/or aged RPE cell that has not been subject to the methods described herein), comprising introducing into the preexisting RPE cell one or more transcription factors selected from the group consisting of OTX2, FOXG1 , Pax6, Smad6, Lhx2, HNF4a, PKNOX2, Sox9, FoxDl, MITF, Mir200, Mir204, Mir211, Klf4, c-myc, Kif9, RARa, Pax2, Smad3, Sox4, Soxl 1, Otxl, Pitx2, Nr2fl, Nr2f2, Tfec and any combination thereof.

In the in vivo methods described herein, introducing a transcription factor of this invention into the cell of the eye can comprise genetically modifying the cell of the eye with one or more nucleic acid molecules comprising a nucleotide sequence encoding one or more transcription factors of this invention. Methods of introducing nucleic acid molecules into the eye can be carried out as described herein and as known in the art.

In the methods described above, the cell in the eye of the subject can be, but is not limited to, a fibroblast, a retinal neuron, an RPE cell, a PR cell, a Mueller glia cell and any combination thereof.

A composition of this invention can be administered to a subject at or near a treatment site in the eye.

The disclosures of all patents, patent publications and non-patent documents cited herein are incorporated herein by reference in their entirety.

The present invention is illustrated in the following non-limiting examples.

EXAMPLES EXAMPLE 1.

The studies describe herein have led to the identification of a set of 13 defined factors (DFs) [mir200 (e.g., mir200b), mir204, mir211, mitf-D, Smad6, HNF4-a, Sox9, PKNOX2, Pax6, FoxDl, FoxGl, Otx2 and Lhx2] that induces mouse embryonic fibroblasts (MEFs) into putative RPE (induced RPE (iRPE)) (n=18 out of 18 wells from 4 independent experiments). This direct conversion protocol using DFs can be realized for RPE replacement, leading to a safe treatment for atrophic ARMD.

Using RPE reporter MEF (RPE65^{/ocZ +}) in studies as described herein, it has been demonstrated that a pool of 13 DFs is sufficient to induce fibroblasts into iRPE. It is likely that not all of these 13 DFs are necessary for iRPE reprogramming. Direct reprogramming approaches are a paradigm shift in regenerative ophthalmology and could pave the way for safe, efficient autologous induced retinal pigment epithelium (iRPE) cell transplantation and in vivo retinal repair. Successful direct RPE induction will provide a roadmap for retinal rehabilitation in patients with atrophic macular degeneration who currently have no FDA-approved treatment.

Photoreceptor loss is the most common cause of irreversible vision loss worldwide. RPE dysfunction and cell death is the cause of photoreceptor loss in non-neo vascular (atrophic) age-related macular degeneration (ARMD). RPE lacks innate regenerative capacity leading to irreversible vision loss after RPE loss. No satisfactory treatment exists for patients with advanced acquired or inherited macular degeneration. Cell-based therapy and direct reprogramming approaches offer hope for patients who have RPE dysfunction, but not significant photoreceptor atrophy.

Current differentiation protocols do not exclusively select for a specific cell-type, and safety concerns have arisen about the possibility of unintentionally transplanting a progenitor cell or having a presumed differentiated cell revert back to a proliferating pluripotent stem cell, possibly developing a tumor. Furthermore, the process of skin biopsy, viral transduction of fibroblasts, stochastic reprogramming with low efficiency, iPSC clone selection (some iPSC or embryonic stem cells (ESCs) clones may have difficulty differentiating into RPE), and RPE purification and differentiation is lengthy and time-inefficient, making clinical implementation difficult. RPE differentiation from iPSCs can take three months or more. This protracted time frame is unacceptable for patients with a narrow window of optimal therapeutic potential. Finally, epigenetic memory and heterogeneity between iPSC clones have resulted in some clones having a differentiation bias for specific lineages, i.e., a neural lineage over a mesodermal lineage. These concerns have prompted a search for a quicker, more efficient method to convert one cell type directly to a desired cell type (bypassing the pluripotent intermediary) that would alleviate safety concerns about accidental stem or progenitor cell transplantation.

A direct reprogramming strategy, with lineage-specific transcription factors, would be more efficient than current reprogramming methods and devoid of tumorigenic transcription factors. The studies described herein are designed to elucidate a paradigm that permits direct conversion of pre-existing cells into iRPE cells for retinal repair, and to provide the foundation for promising therapies for patients with advanced inherited or acquired retinal degeneration. If successful, clinical trials similar to those completed for ESC- and iPSC-RPE could be conducted with iRPE. Also, it may be possible to reprogram dysfunctional RPE into functional RPE using only gene therapy, since direct reprogramming approaches work in post-mitotic cells, unlike with iPSC reprogramming.

This research can be translated to patients with both acquired and inherited RPE degeneration. Patients with acquired RPE degeneration (i.e., atrophic macular degeneration) could benefit from autologous iRPE cell transplantation prior to subfoveal photoreceptor atrophy. Advances in non-invasive imaging such as fundus autofluorescence, spectral domain-optical coherence tomography (SD-OCT), and microperimetry allow for precise measurement of the health and function of the RPE and photoreceptors. High-risk patients would be identified through clinical monitoring and offered a minimally invasive skin biopsy, which would serve as source of fibroblasts. Further investigation could lead to the use of peripheral blood cells to generate iRPE, which would be more convenient and less invasive for patients. A subject's cells would be infected (non-integrating viruses, protein extract, non- viral minicircles, nanoparticle gene delivery) with a cocktail of factors to convert these cells into iRPE. It is also contemplated that the essential defined factors (DFs) may be replaced with small molecules. Regardless of the method, iRPE could be injected in the subretinal space similar to the current method used for ESC-derived RPE in clinical trial. Pluripotent-derived RPE cells are being evaluated in clinical trial; we envision that these cells could prove to be safer and more stable than current cells. Patients with inherited RPE degeneration, such as Stargardts and Bests disease, could benefit from a combination of iRPE with gene replacement therapy in vitro prior to iRPE transplantation. Finally, subjects with advanced neovascular ARMD commonly develop a fibrous (disciform) scar that could serve as an in vivo source of fibroblasts, in addition to reprogramming endogenous diseased RPE.

The present invention provides an innovative approach towards solving the fundamental problem of replacing damaged cells in the retina by directly reprogramming fibroblasts into iRPE for the purpose of preserving or restoring vision through cellular reprogramming. This approach has not been realized for RPE cell replacement therapy and provides a game-changing approach to restoring vision. To achieve this objective, a drug- inducible system is employed to regulate transgene expression coupled with an RPE-specific promoter. Computer modeling is used to identify putative factors that are predicted to bind to regulatory elements in key known RPE genes. Taken together, this approach has led to compelling preliminary data demonstrating that molecular and functional iRPE could be converted directly from fibroblasts, bypassing the current pluripotent stem cell requirement.

Preliminary studies have identified a 13 DF pool that permits conversion of mouse embryonic fibroblasts (MEF) into iRPE. This conclusion is based on the following preliminary data: morphology switch from a long, slender fibroblast shape to a small, round, cuboidal RPE-Iike shape, RPE65 reporter gene expression in trans -differentiated fibroblast cells, a gene expression profile resembling RPE, and functional activity like RPE in the fluorescent latex bead phagocytosis assay, a cardinal RPE function. Experiments have also been carried out with an 8 DF combination and cell morphology switching, a gene expression profile resembling RPE, and RPE65 reporter gene expression were demonstrated.

Selection of candidate factors for iRPE conversion. DFs that are highly expressed in RPE were identified first. A reduced-bias approach was used by cross-referencing master regulators of RPE with Matlnspector software. The software was used to predict which transcription factors could theoretically bind to the promoter region of known key master regulators of RPE specification. Since fibroblasts (mesenchyme) and RPE (neuroepithelium) are from different embryonic lineages, factors thought to be important to promote

mesenchyme to epithelial transition (MET) were added. Based on this analysis, a pool of 13 DFs were screened for cell fate conversion to iRPE.

Tetracycline-On-inducible system with RPE65 reporter. A drug-inducible reporter system was used for the transcription factors to regulate transgene expression in response to dox. For this system to operate, a cell requires both reverse tetracycline-dependent transactivator (rtTA) and a tetracycline (Tet-On) response element. The purpose of utilizing a regulated transgene expression system is to distinguish the role of endogenous epigenetic and genetic changes and to assess stability of iRPE after reprogramming by eliminating exogenous input.

DF plasmids were obtained from Addgene and Origene, requested from other laboratories, or cloned in the laboratory. A commercial collaborator cloned cDNAs into a lenti-virus construct with Tet-On response element driving a ubiquitous promoter (Cellomics, Inc.). M2rtTA transgenic mice (Jackson Labs, #006965) that carry rtTA in the

Gt(ROSA)26Sor locus and constitutively express rtTA in all cells, were used. While lenti- rtTA virus was analyzed, a stronger transgene activation was demonstrated using rtTA transgenic mice. Since one of the 13 DFs is not in an inducible vector, it will be cloned into the same Tet-On vector used for the other DFs and will be used for future experiments. For human experiments, we used lenti-rtTA virus that constitutively expresses rtTA.

To establish an RPE reporter system, we crossed ROSA26^{rtTA rtTA} with RPE65^,ac2'⁺ (NEI). RPE65 is an isomerohydrolase that plays a critical role in the visual cycle by regenerating visual pigment necessary for photoreceptor-mediated function, and is highly specific to RPE, making it a specific RPE reporter. Also, this reporter system can be used to prospectively identify iRPE in a similar fashion to fluorescent reporters using the DetectaGene Green CMFDG lacZ Gene expression kit, a technique we have successfully performed in our laboratory.

El 2.5 day embryos were harvested and digested from pregnant female ROSA26^{rtTA rtTA}; RPE65^{lacZ +} mice, and plated for tissue culture using published methodology (26). Resultant MEFs were transduced with lenti-Tet-On. Dox (2 μg/ml) was used to induce transgene expression in inducible DFs. The dox system was tested for leakiness using a lenti-Tet-On- GFP virus, the same vector backbone used for the other transgenes. GFP expression correlated well to dox exposure.

13 DF can convert MEF into RPE65-positive cells. Passage one ROS A26^rtTA

r^tTA;RPE65^{IacZ +}MEFs were plated at 1 ^χΐθ⁵ cells/well on gelatin-coated plastic culture dishes in MEF growth medium (DMEM plus 10% fetal bovine serum (FBS)). The next day, 13 DF concentrated lenti-viruses and 4 §/ιη1 polybrene (Sigma) were incubated with the cells at a multiplicity of infection (MOI) of 10. Twenty-four hours post-infection, the viral mix was exchanged for fresh MEF growth medium. Forty-eight hours after infection, transduced MEFs were cultured in RPE medium (GMEM plus 5% FBS, 5% KSR, 0.1 mM NEAA, 1 mM sodium pyruvate, 0.1 mM β-mercaptoethanol). Dox was included in the RPE medium for 3 weeks and then withdrawn for at least 1 week.

MET was noted in a subset of cells 5 days after dox induction. After 1 month, positive lacZ (RPE65) expression was found in some epithelial cells, immunofluorescence iRPE staining was positive for BESTROPHIN, CRALBP, and RPE65, anti^-galactosidase (to confirm specificity of RPE65 expression) protein, which are mature RPE markers. EdU analysis revealed that iRPE are proliferating. qPCR revealed up-regulation of RPE signature genes ranging from 2.5- to 200-fold higher than MEF (Fig. 3). This was repeated 4 times in 18 wells, with a reprogramming efficiency of 3.87 ± 0.66%. This efficiency is consistent with direct conversion methods to other cell types (39), and is over 350% more efficient than iPSC reprogramming as initially reported. Also, 13 DF iRPEs have been passaged and expanded by manually picking up clones using a similar method to what has been done with iPSCs.

13 DF can convert human fetal lung fibroblasts (HFL) into epithelial like cells. HFLF cells (American Type Culture Collection) were transduced with a lenti- irus constitutively expressing rtTA and the same 13 DF cocktail we used in these mouse studies (the DFs are highly conserved between mouse and human) at MOI of 10. HFLF were cultured in ATCC- formulated F-12K medium with 10% FBS. After 1 week, the medium was switched to the RPE medium described previously. Twenty-five days after dox induction, a cluster of cells with an altered morphology was noted (Fig. 4A). qPCR revealed up-regulation of RPE signature genes ranging from 3.5- to 4000-fold higher than HFLF control (Fig. 4B).

8 DF can convert MEF into RPE65~positive cells. Experiments have been carried out in which the number of DF is reduced from 13 DF to 8 DF [mir200 (e.g., mir200b), mir204, mir211 , Mitf-D, Otx2, Sox9, FoxDl and Pknox2]. 8 DF cells showed a cuboidal, epithelial morphology similar to 13 DF iRPE. After 1 month of dox followed by 2 weeks of dox withdrawal, the iRPE cells were RPE65-positive by X-gal staining in contrast to the non- transformed MEF. The conversion efficiency of 8 DF was higher than 13 DF (7.82 ± 0.79% vs. 3.87±0.66%). An 11 DF combination [mir200 (e.g., mir200b), mir204, mir21 1, Otx2, Mitf-D, FoxD 1 , Sox9, P NOX2, HNF4-aIpha, and LHX2] also successfully converted fibroblasts to iRPE with an efficiency of 6.58 ± 0.71%. These data suggest that 8 DF or fewer factors may be sufficient for iRPE generation, and could have advantages over 13 DF. Also, certain 1 , 2, and 3 DF combinations have been tried where MET was absent at 2 weeks, suggesting that certain combinations of 1 , 2 or 3DF may not generate iRPE under the conditions described herein.

iPSC-derived RPE and native adult mouse RPE will be used as a control. Mouse iPSCs were generated using a previous protocol. Mouse iPSCs were grown as embryoid bodies, and then switched to retinal differentiation media (DMEM/F12 Glutamax containing N2 supplement and Pen/strep) and transferred into low-binding plates similar to the method of Gonzales-Cordero et al. After 2 days, aggregates were allowed to attach to the culture dish with the addition of laminin. To allow for RPE differentiation, the medium for the iPSC- derived neuroepithelial rosettes was switched to a chemically defined RPE differentiation medium consisting of DMEM/ 12 (3:1) supplemented with 2% B27 on day 16 of

differentiation. iPSC-derived RPE will be used a control for immunofluorescence and qPCR studies.

13 DF demonstrate in vitro functionality in a phagocytosis assay. 13 DF MEF underwent dox induction for 28 days, resulting in iRPE, followed by dox withdrawal for 2 weeks.

Stable iRPE was incubated with fluorescent latex beads to perform the phagocytosis assay. A primary function of native RPE is phagocytosis of shed photoreceptor outer segments. RCS rats with a Mertk mutation are not able to clear these shed outer segments, resulting in extracellular accumulation in the subretinal space. To determine if iRPE cells have phagocytosis capability, cells were incubated with 1 μΜ of fluorescent latex beads

(Invitrogen) for 16 hours, washed with PBS, dissociated with trypsin, and then plated on Lab- Tek chamber slides. iRPE was co-stained with anti-RPE65 and anti-CRALBP. Confocal microscopy z-stack demonstrated intracellular fluorescent beads in RPE65- and CRALBP- positive cells.

State-of-the-art imaging can be used to monitor photoreceptor degeneration, SD-OCT is a non-invasive imaging modality that measures reflectance of light using low-coherence interferometry to provide cross-sectional images of the retina comparable to histology. Full- field or global ERG measures retinal electrical activity in response to light stimulation, but requires approximately 150,000 rod photoreceptors, a significant amount of functioning photoreceptors. Image-guided focal ERG (Micron III and IV, Phoenix Laboratories) allows stimulation of a specific region of the retina (the location of inj ected cells in Aim 2B) with real-time comparison to another region of retina (a location where no cells were injected) in the same eye by moving a red aiming beam. The ability to conduct longitudinal studies reduces the variability that can occur with litter and species comparisons, providing increased confidence in interpreting results compared to the standard technique of group comparison.

In vivo reprogramming is an exciting focus of regenerative medicine. Knowledge gained from studies described above will allow for studies to analyze a discrete set of DFs for reprogramming transplanted or preexisting retinal cells in vivo. The benefit of this method is the ability to use the retina microenvironment to guide iRPE maturation and improve function or possibly avoid transplantation of cells altogether by transducing and trans-differentiating preexisting cells.

In vivo reprogramming of exogenous cell transplantation may have advantages over traditional cell replacement therapy. Reprogramming in vivo allows the opportunity to gain instructional cues from the retinal microenvironment, possibly aiding and improving cell migration, polarization, and function, while also overcoming epigenetic roadblocks. Many retinal degenerations lead to RPE atrophy, such as atrophic ARMD, where there are no endogenous cells that can be used for iRPE conversion, requiring exogenous cell

transplantation.

In vivo reprogramming of endogenous cells with DFs could avoid the need for cell transplantation. Certain retinal degenerations, such as neovascular AR D, lead to disciform scar, consisting of fibroblasts, which could be used as an endogenous source of cells for reprogramming. DFs could be delivered to these fibroblasts for cell conversion into iRPE. Another strategy is to reprogram diseased RPE with DFs in an attempt to reprogram them into youthful RPE. While there is no cell fate switching, it may be possible to use a combination of DFs to modify the epigenetic signature that causes aging and RPE

dysfunction. Also, it is important to note that direct reprogramming approaches work in post- mitotic cells unlike methods to reprogram cells into iPSCs, making direct reprogramming methods advantageous for RPE cells that have limited proliferation in normal conditions.

EXAMPLE 2. .Defining the necessary molecular determinants to convert fibroblasts into functional induced retinal cells.

The objective of these studies is to identify the essential transcription factors and non- coding RNAs (collectively referred to as DFs in this proposal) that convert fibroblasts into iRPE. Preliminary studies identified a 13 DF pool that permits conversion of mouse embryonic fibroblasts (MEFs) into iRPE. This conclusion is based on the following preliminary data: morphology switch from a long, slender fibroblast shape to a small, cuboidal RPE-like shape (Fig. 2A), dark brown pigment appearance (Fig. 2A), RPE65 reporter gene expression in trans-differentiated fibroblast cells (Figs. 2B-C), a gene expression profile resembling RPE (Fig. 3), and functional activity using the fluorescent latex bead phagocytosis assay, a cardinal RPE function. While it was determined that 13 DFs are sufficient to convert MEF into iRPE, the expectation is that not all 13 DFs are necessary for reprogramming. To that end, preliminary experiments were performed with an 8 DF combination, which demonstrated cell morphology switching, a gene expression profile resembling RPE, and RPE65 reporter gene expression. Studies are designed wherein the number of DFs is sequentially reduced to identify the essential core of DFs. These detailed molecular analyses will compare 13 DF, minimal DF (the core DFs), native RPE, and iPSC- derived RPE for both mouse and human cells. Both mouse and human experiments are necessary because the essential combination of factors is likely to vary between species. Both 13 DF and minimal DF iRPE will be analyzed in parallel.

Once a DF pool (typically 10-20 factors) is identified that successfully induces a cell morphology change towards the desired cell lineage and the reporter gene is activated, systematic identification of the critical reprogramming factors occurs by excluding one or two genes at a time. This process typically leads to identification of a few critical factors. Detailed molecular characterization, and in vitro, and in some cases in vivo, functional studies are then performed to demonstrate similarity to the native cell of interest.

Transient induction of pluripotent genes produces RPE-like cells. Transient induction of

4F (e.g., Sox2, lf-4, c-Myc, Oct3/4) has been used to partially reprogram cells in vitro, bypassing a stable pluripotent intermediary. Fibroblasts have been converted into neural progenitor cells and cardiomyocytes using this method. Using a similar approach has allowed for the generation of putative iRPE, bypassing pluripotency (iPSCs). The commercially available mouse harboring a tetracycline-inducible transgene (called 4F2A mice herein) _(ROSA26^rtTA;tet0n-^!>ou5fl'-^Sox2'^'Klf '^"Myc(4F2A)) (Jackson Labs, Bar Harbor, ME) was used. The transgene has four pluripotency factors SOX2_y KLF-4, c-MYC, OCT 3/4) regulated by a tetracycline transcription activator (a doxycycline-inducible system). These experiments were performed to determine if fibroblasts could be reprogrammed into RPE- like cells. Adult tail tip fibroblasts from 4F2A mice were treated for 7 days with doxycycline (dox), a tetracycline analogue. Fibroblast media were switched to neural induction media for 6 days followed by 12 days of RPE growth medium. RPE-like cells emerged 3 weeks after initiating this protocol. The molecular characterization of partial iRPE demonstrated many similarities to RPE-like cells. qPCR experiments revealed increased expression of TJPl(ZO- 1), PEDF, RBLP (CRALBP), MITF, OTX2, and VMD2 as compared to fibroblasts.

Immunofluorescence was positive for ZO-1. Electrophysiology testing was also carried out, which was supportive of a Na⁺/K⁺ pump, consistent with an RPE identity. While this work bypassed a pluripotent intermediary, and would significantly reduce the time needed to generate RPE from approximately 16 weeks with iPSC to 4-6 weeks, it did require differentiation with neural induction media and likely resulted in immature RPE similar to that produced by iPSC and ESC methods. As a result of this work, it was determined that a transdifferentiation protocol could be realized for generating RPE replacement cells with defined factors.

Candidate factor selection for iRPE conversion. DFs highly expressed in RPE were identified through transcriptome analysis of authentic RPE. Unique gene expression profiles were identified between embryonic, fetal and adult RPE. A reduced-bias approach was developed by cross-referencing master regulators of RPE that were highly expressed in both development and mature RPE with Matlnspector software. This software was used to identify which transcription factors could theoretically bind to the promoter region of known key master regulators of RPE specification. As fibroblasts (mesenchyme) and RPE

(neuroepithelium) are from different embryonic lineages, the DFs mir200b, mir204, and mir211 were added, all of which block epithelial-to-mesenchyme transition and therefore indirectly promote mesenchyme-to-epithelial transition (MET), and are highly expressed in mature RPE. The transcription factors FoxDl, Pknox2, and Smad6 were considered because they are highly expressed in mature RPE. Based on this analysis, a pool of 13 DFs were screened for cell fate conversion to iRPE. The 13 DFs were the following: mir200b, mir204, mir2U, MITF-D, OTX2, SOX9, FOXD1, PKNOX2, PAX6, SMAD6, FOXG1, HNF4-a, and LHX2 (Fig. IB). Tetracycline-On-inducible system with RPE65 reporter. A drug-inducible reporter system was used for these transcription factors to regulate transgene expression in response to dox. For this system to operate, a cell requires both a reverse tetracycline-dependent transactivator (rtTA) and a tetracycline (Tet-On) response element. The purpose of utilizing a regulated transgene expression system was to distinguish the role of endogenous epigenetic and genetic changes, and to assess stability of iRPE after reprogramming by eliminating exogenous input.

cDNAs encoding the DFs were cloned into a lenti-virus construct with Tet-On response element driving a ubiquitous promoter (Cellomics, Inc.). M2rtTA transgenic mice (Jackson Labs, #006965) were used, which carry rtTA in the Gt(ROSA)26Sor locus and constitutively express rtTA in all cells. For human experiments, lenti-rtTA virus that constitutively expresses rtTA was used.

To establish the mouse RPE reporter system, ROSA26^{rtTA rtTA} was crossed with

RPE65^lacZ/÷ (NEI). RPE65 is an isomerohydrolase that plays a critical role in the visual cycle by regenerating visual pigment necessary for photoreceptor-mediated function, and is highly specific to RPE, making it a specific RPE reporter. This reporter system can be used to prospectively identify iRPE in a similar fashion to fluorescence reporters using the

DetectaGene Green CMFDG lacZ Gene expression kit. To determine if iRPE resulted through transdifferentiation or spontaneous differentiation through a PAX6⁺ progenitor state ROSA26^{rtTA TA}; RPE65^iacZ were crossed with PAX6^EGFP mice. PAX6^EGFP expression was confirmed in the adult neurosensory retina of these mice. This allowed for longitudinal assessment of PAX6 expression in cell culture during the reprogramming phase of the experiments. This reporter is important to distinguish iRPE from generic epithelial cells or other pigmented cell types.

E12.5 day embryos were harvested and digested from pregnant female ROSA26^rtTA/ ^rtTA;RPE65^iac2;+ or ROSA26^rtTA/rtTA;RPE65^,ecZ/+;PAX6^EGFPmice, and plated for tissue culture. Resultant MEFs were transduced with lenti-Tet-On-DFs. Dox (2 μg/ml) was used to induce transgene expression in inducible DFs. The dox system was tested for leakiness using a lenti- Tet-On-GFP virus, the same vector backbone used for the other transgenes. GFP expression correlated well to dox exposure.

13 DF can convert MEF into RPE65 -positive cells without transitioning through a PAX6 progenitor state. Passage one ROSA26^{rtTA rtTA};RPE65^{iacZ +};PAX6^EGFP MEFs were plated at 1 x 10⁵ cells/well on gelatin-coated plastic culture dishes in MEF growth medium (DMEM plus 10% fetal bovine serum (FBS)). The next day, 13 DF concentrated lenti-viruses and 4 μg/ml polybrene (Sigma) were incubated with the cells at a multiplicity of infection (MOI) of 10. Twenty-four hours post-infection, the viral mix was exchanged for fresh MEF growth medium. Forty-eight hours after infection, transduced MEFs were cultured in RPE medium (GMEM plus 5% FBS, 5% KSR, 0.1 mM NEAA, 1 mM sodium pyruvate, 0.1 ιηΜβ- mercaptoethanol). Dox was included in the RPE medium for 3 weeks and then withdrawn for at least 1 week.

MET was noted in a subset of cells 5 days after dox induction. Since MET is known to be an essential step in iPSC reprogramming, studies were done to determine if the resultant cells were generic epithelial cells or a retinal sub-type. Also, while the cells appeared pigmented, the possibility was considered that these cells could be melanocytes. After one month of cell culture, X-gal staining was performed, demonstrating positive lacZ (RPE65) expression, a specific RPE marker, in some epithelial cells (Fig. 2B). Also, longitudinal analysis using 13 and 12 DF combinations (Fig. 2B) did not reveal PAX6-EGFP expression over 1 month, suggesting that these cells did not transition through a PAX6 progenitor state supporting the claim that this is a transdifferentiation process (Fig. 1 A). As a positive control, PAX6-EGFP expression was confirmed in the adult neurosensory retina and in cell culture. After 1 month, qPCR was performed 1 week after dox withdrawal and revealed up- regulation of RPE signature genes ranging from 2.5- to 370-fold higher than MEF (Fig. 3A). Otx2 and Lhx2 were in constitutive vectors so qPCR experiments were repeated with inducible Tet-On Otx2 and without Lhx2 using the same 3 weeks of dox followed by 1 week of dox withdrawal (12 DF; mir200b, mir204, mir21l, MITF-D, OTX2, SOX9, FOXD1, PKNOX2, PAX6, SMAD6, FOXG1, HNF4-a). A similar gene expression profile was noted (Fig. 3B). An 8 DF combination, without any microRNAs (Gapdh, Otx2, Mitf, Sox9, Foxdl, Pknox2, Hnf4-a, Lhx2, Six6 and Six3), demonstrated genotypic and phenotypic stability without dox for 6 weeks (Fig. 3C (E^.S^embryo). iRPE immunofluorescence staining was positive for MITF, BESTROPHIN, CRALBP, ZO-1, and RPE65, which are mature RPE markers. Protein expression, in contrast to gene expression, of RPE65 suggests maturity, and is often not found in iPSC-RPE. EdU analysis revealed that iRPE can proliferate but further study has shown that this proliferation is limited. EdU analysis was repeated 4 times in 18 wells, with a reprogramming efficiency of 3.87 ± 0.66%.

13 DF can convert human fetal lung fibroblasts (HFLF) into putative iRPE cells. HFLF (American Type Culture Collection) were transduced with a lenti-virus constitutively expressing rtTA and the same 13 DF cocktail used in the mouse studies at an MOI of 10. (The DFs are highly conserved between mouse and human.) HFLF were cultured in ATCC- formulated F-12K medium with 10% FBS. After 1 week, the medium was switched to the RPE medium described previously. Twenty-five days after dox induction, a cluster of cells with an altered morphology was noted (Fig. 4A). qPCR revealed up-regulation of RPE signature genes ranging from 3.5- to 4000-fold higher than HFLF control (Fig. 4B).

8 DF can convert MEF into RPE65 -positive cells. The 8 DF combination in Fig. 2 was comprised of 3 microRNAs and 5 transcription factors: mir200, mir204, mirlll, M1TF-D, OTX2, SOX9, FOXD1, and PKNOX2. 8 DF cells showed a cuboidal, epithelial morphology similar to 13 DF iRPE (Fig. 2C). After 3 weeks of dox followed by 2 weeks of dox withdrawal, the iRPE cells were RPE65-positive by X-gal staining in comparison to the non- transformed MEF (Fig. 2C). The conversion efficiency of 8 DF was higher than 13 DF

(7.82±0.79% vs. 3.87±0.66%). An 11 DF combination (mir200, mir204, mir211, Orx2, Mitf- D, FoxDl, Sox9, PKNOX2, HNF4-alpha, and LHX2) also successfully converted fibroblasts to iRPE with an efficiency of 6.58±0.71%. These data suggest that 8 DF or fewer factors may be sufficient for iRPE generation. Additionally, the small molecules valproic acid, CHIR99021, tranylcypromine, and forskolin are being used to replace 7 of the DFs (6DFs + small molecules). qPCR analysis showed a 903-, 529-, and 203-fold increase in RLBP, TYR, and ZO-1, respectively.

iPSC-derived RPE and native adult mouse RPE will be used as a control. Mouse iPSCs were grown as embryoid bodies, and then switched to retinal differentiation media

(DMEM/F12 Glutamax containing N2 supplement and Pen/strep) and transferred into low- binding plates. After 2 days, aggregates were allowed to attach to the culture dish with the addition of laminin. To allow for RPE differentiation, the medium for the iPSC-derived neuroepithelial rosettes was switched to a chemically defined RPE differentiation medium consisting of DMEM/F12 (3 : 1) supplemented with 2% B27 on day 1 of differentiation (Fig. 2D). iPSC-derived RPE will be used a control for immunofluorescence and qPCR studies.

To identify the essential DFs that permit fibroblast conversion and to provide insight into the hierarchical regulatory network resulting in iRPE, a systematic approach will be employed, of sequentially reducing the DF pool by one DF at a time. Current shows that only 8 DFs are necessary to generate RPE65^!acZ-positive cells. It is expected that fewer DFs (possibly 4 or 5) are sufficient for iRPE conversion, and that reduced DF iRPE functions similarly to 13 DF iRPE.

Identifying the core group of DFs for iRPE conversion. Using the reporter system described herein, transduce ROS A26^{rtTA/ rtTA}; RPE65^{lacZ +} MEF will be transduced with each lenti -virus transcription factor at an MOI of 10, systematically reducing DFs by 1 or 2 factors at a time. Cell culture experiments will be carried out in triplicate in a seeding density of 1 x 10⁵ MEFs in 48 well plates. 13 DF will serve as a positive control for each experiment. Cell morphology switching to an epithelial shape (mesenchymal-epithelial transition; MET) will be assessed as the first step towards an iRPE fate. In data previously described, it was noted that the transition occurs within 5 to 7 days after dox induction in MEF. Therefore, if we MET is not observed by about 2 weeks after dox induction, it may be assumed that the combination of DFs was not sufficient for iRPE conversion. DF combinations that reveal an epithelial morphology switch will be cultured in dox for at least 21 days.

To determine if these cells express RPE65, the DetectaGene Green CMFDG lacZ Gene Expression Kit (Invitrogen) will be used according to the manufacturer's protocol to have lacZ-positive cells fluoresce in the green wavelength. If negative, these cells will be cultured with dox for an additional 2 weeks and will undergo X-gal staining (Sigma), which has less background than CMFDG. These experiments will determine the core group of factors necessary for iRPE conversion. To determine if this core group requires ectopic DF expression for iRPE maintenance, dox will be withdrawn for at least 7 days to identify genetically and phenotypically stable iRPE. 13 DF and the combination with the least number of DFs (minimal DFs) will undergo the detailed molecular and RPE authenticity analysis described herein. Time course experiments with 13 DF and minimal DF will be conducted, using the DetectaGene Green CMFDG lacZ Gene Expression Kit, by adding CMFDG substrate every day to determine when RPE65 expression can first be detected. Preliminary data suggest that RPE65 expression occurs between day 14 and 21 after dox. After these criteria are met, ROSA26^rtTA/rtTA;RPE65^lacZ;PAX6^EGFP mice without CMFDG will be used to rule out the possibility that minimal DF undergo differentiation through a PAX6 progenitor state.

Systematic reduction of DFs will be carried out in human fibroblasts once in vitro analyses have been performed on 13 DF iRPE as described in the data for mice. No reporter will be used for human cell experiments as a pigmented, honeycomb cell morphology switch will be used as an indicator of iRPE cell conversion. PAX6^luc reporter (Qiagen, Valencia, CA) will be used to rule out a PAX6 progenitor state.

Reprogrammed 13 DF and minimal DF from the studies above will be purified, expanded, and devoid of non-reprogrammed fibroblasts for molecular analysis and in vitro and in vivo characterization. A mouse reporter system has been chosen to provide the ability to perform FACS. It is expected that 13 DF and minimal DF mouse iRPE can be cultured and expanded devoid of fibroblasts since they demonstrate properties of clonal expansion in cell culture. Similarly, it is expected that human colonies can be handpicked to enrich the human iRPE population. Purified mouse and human iRPE will undergo

immunohistochemistry, bi-sulfite sequencing, electron microscopy, and qPCR.

iRPE purification and characterization. After dox withdrawal for 1 week to select for genotypically stable cells, 13 DF and minimal DF iRPE will undergo FACS analysis and capture. FluoReporter lacZ (Invitrogen) will be used to fluoresce / cZ-positive cells, Dox- nai^'ve, virus -infected MEF and GFP-positive cells will be used as negative and positive controls, respectively. FACS-captured mouse iRPE will be cultured and expanded. For human iRPE, an inverted microscope will be used to pick up iRPE colonies. The purpose is to generate a phenotypically stable, expandable population of iRPE for characterization and in vivo studies. The following characterization studies will be performed to determine the resemblance of iRPE to native RPE and iPSC-RPE.

IHC/IF studies will include antibody staining for PAX6, RPE65, CRALBP, BEST1, OTX2, MITF, RLBP1 using standard techniques. CLAUDIN-3 localization to the tight junction, and ZOl and CLAUDIN- 19 to the lateral membrane will be examined by confocal microscopy for assessment of the outer retinal blood barrier. To determine if iRPE have apical localization of Na⁺/K⁺ ATPase, we will perform IF studies with anti- Na^{+ +} ATPase a-1 (Upstate Biotech, Charlottesville, VA) (44,67).

Quantitative RT-PCR gene expression studies will include 154 RPE signature genes, some of which are CRALBP, VMD2, MERTK, MITF, OCLN, RPE65, TYR, TYRP1, TJP1 (ZO-1), PEDF, VEGFA, PMEL-17, OTX2, PAX6, and phagocytic genes MERTK, LAMP2, VDP, and GULPL RPE maturity will also be measured by N-cadherin, E-cadherin, claudin, and occludin expression. iPSC-RPE and fibroblasts will serve as controls.

Electron microscopy will be performed. Briefly, enriched mouse and human 13 DF and minimal DF will be seeded on gelatin-coated transwells, and then fixed in arnovsky's fixative (1% paraformaldehyde and 3% glutaraldehyde in 0.1 M cacodylate buffer). Cells plus membrane will be excised from the transwell and will undergo post-fixation and dehydration through a graded series of alcohols and epoxypropane. Studies will be done to search for features characteristic of RPE: cuboidal shape, apical microvilli, melanasomes, and adherens junctions.

Bisulfite sequencing for CpGs analyzed in the promoter region of RPE65 and VMD2 will be performed. The purpose of bisulfite sequencing is to determine if ectopic DF expression leads to an epi genetic profile mimicking native RPE in comparison to fibroblasts.

Demethylation of these promoter regions, with silenced transgene expression (no dox), suggests that transient, ectopic DF expression led to epigenetic changes that promoted activation of key RPE genes, a cardinal feature of cellular reprogramming. Fibroblasts and iPSC-RPE will be used for comparison, as well as primary native adult and fetal mouse and human RPE.

EXAMPLE 3. Determination if iRPE can serve a similar function to native RPE in vitro and in vivo,

Stable iRPE was incubated with fluorescent latex beads to perform the phagocytosis assay. A primary function of native RPE is phagocytosis of shed photoreceptor outer segments. RCS rats with a Mertk mutation are not able to clear these shed outer segments, resulting in extracellular accumulation in the subretinal space. To determine if iRPE cells have phagocytosis capability, 1 μΜ of fluorescent latex beads (Invitrogen) was incubated with cells for 16 hours; cells were washed with PBS, dissociated with trypsin, and then plated on Lab-Tek chamber slides. iRPE was co-stained with anti-RPE65 and anti-CRALBP.

Confocal microscopy z-stack demonstrated intracellular fluorescent beads in RPE65- and CRALBP-positive cells.

State-of-the-art imaging can be used to monitor photoreceptor degeneration. SD-OCT is a non-invasive imaging modality that measures reflectance of light using low-coherence interferometry to provide cross-sectional images of the retina comparable to histology (Fig. 5). Full-field or global ERG measures retinal electrical activity in response to light stimulation, but requires approximately 150,000 rod photoreceptors, a significant amount of functioning photoreceptors. Image-guided focal ERG (Micron III and IV, Phoenix

Laboratories) allows stimulation of a specific region of the retina (the location of injected cells) with real-time comparison to another region of retina (a location where no cells were injected) in the same eye by moving a red aiming beam. The ability to conduct longitudinal studies reduces the variability that can occur with litter and species comparisons, crucial for providing increased confidence in interpreting results compared to the standard technique of group comparison.

Transient induction of 4F using a double transgenic mouse (4F2A), Proof-of-concept experiments have been carried out to determine if partial 4F induction can convert a preexisting retinal cell into a photoreceptor precursor that replaces dead photoreceptors in the setting of photoreceptor injury. Yamanaka factors are capable of switching cell identity. To transiently induce Oct 3/4, Sox2, Klf-4, and c-Myc, a double transgenic mouse line with a doxycycline-inducible polycistronic 4F2A cassette was used, containing all 4F [(ROSA26 ^TA; Collaltm3 ^^10* oco^/i^-n^if^cMycj _{(Jackson bs})] _{to 4F} transgene expression. All cells in this 4F2A mouse expressed 4F in response to dox exposure. To test the fidelity of this system, ciliary body cells and tail-tip fibroblasts were dox-induced in vitro, which were able to generate iPSCs to confirm 4F-mediated cellular reprogramming. Also, tail-tip fibroblasts were dox- induced for 3-7 days, partially reprogramming them into a de-differentiated state without generating iPSCs. Culture conditions were switched to generate putative retinal pigment epithelial cells in vitro. These experiments demonstrated that partial 4F induction could partially reprogram a fibroblast into a retinal cell type given specific culture conditions that facilitate retinal specification. This success led to trying an in vivo reprogramming approach using this transgenic mouse.

Systemic dox induces retinal transgene expression. Three routes of doxycycline administration were tested-gastric lavage, SC, and intravitreal dox-and retinal transgene expression was measured by all routes of administration.

4F-induced retinal rehabilitation after MNU injury. N-Methyl-N-nitrosourea (MNU) is a DNA-alkylating chemotherapeutic agent that causes photoreceptor cell death by apoptosis within 4-7 days. A single intraperitoneal injection caused irreversible photoreceptor apoptosis in more than 95% of photoreceptors (Figs. 5E-F). Long-term studies (>2 months) did not demonstrate spontaneous regeneration.

With the following protocol retinal repair was detected in a small subset of mice (n-4 out of 24 eyes in 2 independent experiments): One SC dox injection followed by 1

intraperitoneal injection of MNU 5 days afterwards. SD-OCT was used and measured an increase in total retinal and outer retinal thickness in 4F+MNU (Fig, 5H) compared to MNU only (Fig.5E). Longitudinal full-field ERO studies were performed in the same eye over time to confirm near total ablation of the outer retina and loss of ERG amplitudes by day 7, followed by subsequent gradual improvement in retinal electrical response (Fig.6). Full- field ERG revealed nearly extinguished electrical amplitudes consistent with the SD-OCT findings (Fig.6A, middle) after MNU only. In four 4F+MNU eyes, but never in MNU-only mice, regeneration of the outer nuclear layer was noticed on SD-OCT. In two 4F+MNU eyes, time-dependent improvement in ERG was noticed, up to 80% of normal (Fig.6A, right). Focal ERG improvement was demonstrated in one 4F+MNU eye that did not demonstrate improvement on full-field ERG. New photoreceptor-like cells in the outer retina

42 were corroborated by post-mortem histology (Fig. 51). Immunofluorescence studies demonstrated the presence of rod and a few cone photoreceptors in 4F+MNU mice. An abnormal appearance of rods and cones was noted compared to WT. There were no photoreceptors in the MNU image.

Measure transepithelial resistance (TER), resting membrane potential, Na^/K^ voltage- gated channels, and Ca²^ imaging in iRPE. To assess the barrier function of iRPE, which is a critical aspect of maturity, mouse and human 13 DF and minimal DF iRPE will be cultured, without dox for at least 1 week, iPSC-RPE, adult and fetal RPE monolayers on transwell filters and TER will be measured using an epithelial voltohmmeter (EVOM2) following the manufacturer's instructions (World Precision Instruments, Sarasota, FL). The TER values obtained from the EVOM2 will be multiplied by the transwell surface area to obtain the true TER measurement.

Whole-cell voltage patch clamp electrophysiology recordings of single RPE cells will be performed to measure resting membrane potential and Na⁺, ⁺, and CY apical -basal permeability of iRPE. iRPE will be plated on poly-D-lysine and laminin-coated coverslips for 1 week. iPSC-derived and fetal RPE will be used as a controls. Cells will be

continuously perfused with solution in a recording chamber, and identified and analyzed using a microscope connected to a CCD camera.

Resting membrane potential will be determined by continuously recording from a patch electrode. To activate voltage-gated currents, cells will be exposed to a voltage step protocol. To determine if iRPE possess potassium channels, 1 μΜ Tetrodotoxin, a sodium channel blocker, will be applied. It is anticipated that application of Tetrodotoxin should leave only potassium currents.

Calcium imaging will be performed on iRPE, iPSC-RPE, and fetal and adult RPE cells incubated with 10 μΜ Fura-2- AM (Calbiochem, San Diego, C A) and 0.02% Pluronic F 127 (BASF, Mount Olive, NJ) for 1 hour at 37°C. Cells will be subsequently washed for 30 minutes and placed in a recording chamber on an inverted microscope. Fura-2- AM emission ratios will be obtained with alternating exposures (340 and 380 nm) using a Photometries Cascade camera. ATP-stimulated changes in intracellular Ca²⁺ will be obtained by exposing RPE cells to 100 μΜ ATP for 2 minutes during recording.

Polarized secretion of VEGF and PEDF assay. Mouse and human 13 DF and minimal DF iRPE, without dox for at least 1 week, will be cultured on transwell membranes for 6 weeks to determine if iRPE have polarized cytokine secretion similar to native RPE. 1 ml of media from the upper and lower reservoirs will be used for analysis performed in triplicate. Mouse and human VEGF and PEDF ELISA will be analyzed.

Retmoid cycling. Mouse and human 13 DF and minimal DF iRPE will be serum-starved for 8 hours followed by a 24-hour incubation with 10 μΜ ail-trans retinol (Sigma- Aldrich, St. Louis, MO) in 2% BS A plus 15% fetal bovine serum. Cells will then be homogenized and intracellular retinoids will be separated using HPLC, and iRPE not incubated with all- trans retinol will be included as a control for endogenous retinoids. Quantification, by comparison of retinoid standard curves (Sigma- Aldrich), of ail-trans retinyl palmitate will be performed using HPLC. To support these findings, lecithin-retinol acyl transferase (LRAT) will be inhibited with 20 μΜ N-ethylmaleimide (NEM) to prevent retinyl ester synthesis and measure all-trans retinyl palmitate.

Photoreceptor outer segment phagocytosis (POS) assay. Confluent, polarized mouse and human 13 DF and minimal DF iRPE will be grown on 24-well clear bottom black plates. After aspirating RPE medium, iRPE will be incubated with FITC-labeled porcine POS at a concentration of 10 POS/iRPE for 2 hours. To terminate the POS challenge, samples will be thoroughly rinse with PBS supplemented with CaCl₂ and MgCl₂ (PBS-CM). Two sets of triplicate samples will be analyzed for separate detection of total and internalized POS quantification. For internalized POS detection, PBS-CM will be aspirated and cells will be incubated with FITC-quenching solution for 10 minutes. For total POS quantification, PBS- CM will remain. All samples will be fixed with ice-cold methanol, and a fluorescence flatbed scanner for FITC detection will be used. Transduced (but not dox exposed) fibroblasts will be used as a control since fibroblasts should not possess POS phagocytosis capability.

Similar to in vitro analyses, it is expected that iRPE will function in the subretinal space similar to native RPE. Experiments will be carried out in RCS rats to determine if iRPE can replace dysfunctional RPE, which leads to photoreceptor dysfunction and poor visual discrimination as a consequence of a Mertk mutation. In this model, RPE dysfunction leads to its inability to phagocytize photoreceptor outer segments resulting in photoreceptor degeneration and vision loss. iRPE comparisons will be made against fibroblast-injected RCS rat eyes to reduce concern of non-specific cell rescue in this model.

To determine if iRPE can prevent photoreceptor degeneration, subretinal injection will be carried out, with about 5x10⁴ of either human or mouse 13 DF and human or mouse minimal DF iRPE cells, which are lenti-CMV-RFP fluorescently tagged for tracking, in RCS rats (n=20 rat eyes for each group (80 eyes)). At the onset of photoreceptor degeneration, iRPE will be injected on P22 in RCS rats. Transduced, dox-nai've MEF will be used as a control in the fellow eye (n^lO MEF-injected). Ten uninjected eyes will serve as a control.

Forty-eight hours prior to the procedure, the rats will receive daily intraperitoneal ciclosporin (10 mg kg) to suppress the immune system and prevent immune rejection of the xenografted iRPE cells. Longitudinal SD-OCT imaging will be performed to quantify outer nuclear layer thickness using a segmentation algorithm included in the SD-OCT software at about 1 week, 1 month, 2 months, and 3 months after transplantation. Micron IV fundus photography with fluorescence filters will be used to detect fluorescently tagged iRPE cells at the previously mentioned time points. Full-field ERG and optokinetic visual discrimination studies will be performed at about 3, 6, 9, 12, and 15 months. Focal ERG will be performed with negative results from full-field ERG in locations where iRPE engraftment is observed by SD-OCT or fundus fluorescence. Eyes demonstrating photoreceptor preservation or visual function will be sectioned and compared to at least 8 dox-naive, fibroblast-injected eyes and 8 uninjected eyes. 6 iRPE injected eyes will undergo retinal flatmount to determine the extent of iRPE engraftment. Engraftment will be determined by performing anti-RFP and anti-human (when human cells injected) antibody and X-gal (for RPE65 expression in mouse cells) to detect the presence and location of fluorescently tagged iRPE. Measurement will be made of c-Fos expression using anti-c-Fos antibody. Light is known to induce c-Fos expression in the inner nuclear layer of the wild-type mouse retina; however, this response is reduced with photoreceptor loss. Adjacent sections will be co-labeled with CRALBP,

BESTROPHIN, and MITF. Hematoxylin and eosin sections will be used to measure outer nuclear layer thickness on 10 sections before and after the optic nerve.

EXAMPLE 4. Retinal Pigment Epithelium (RPE).

The factors listed below were added on mouse embryonic fibroblasts derived from an RPE65-LacZ reporter mouse and a morphologic and gene expression profile change consistent with RPE cells was shown.

Transcription factor pool: OTX2, FOXG1, Pax6, Smad6, Lhx2, HNF4- _} PKNOX2, Sox9, FoxDl, MITF, Mir200b, Mir204, Mir211. Exemplary iRPE Culture Conditions

EXAMPLE 5. Photoreceptor (PR) cells.

The factors listed below were added on mouse embryonic fibroblasts derived from an NRL-GFP reporter mouse and a morphologic and gene expression profile change consistent with PR cells was shown.

Transcription factor pool: Ascll, Crx, Nrl, NeuroDl, Rax, Otx2, mirl24, FoxGl, Pax6,

Ars2.

Exemplary iPR Culture Conditions

EXAMPLE 6. Retinal Progenitor Cells (RPCs).

The factors listed below were added on mouse embryonic fibroblasts derived from an Pax6-GFP reporter mouse and a morphologic and gene expression profile change consistent with RPCs was shown.

Transcription factor pool we tested with all of these combinations as shown by row:

Exemplary iRPC Culture Conditions

1 st Week 2nd Week 3rd Week 4th Week

|G-MEM 86. 82% 89. 82% 90. 82% 91. 82%

FBS 5% 2% 1% 0%

KSR 5% 5% 5% 5%

|NEAA _{. .} 0. Ira M 0. lm 0. lm M 0. lm M

Sodium Pyruvate 1 m M 1 m 1 m M 1 m M

2-Mercaptoethanol 0. lm M 0. lm M 0. lm M 0. lm M

PSF 100 IU/ml 100 IU/ml 100 IU/ml 100 IU/ml

The foregoing is illustrative of the present invention, and is not to be construed as limiting thereof. The invention is defined by the following claims, with equivalents of the claims to be included therein.

Table 1. Human transcription factors

Table 2. Murine transcription factors

Claims

THAT WHICH IS CLAIMED IS:

1. A method of generating an induced retinal pigment epithelium cell, comprising introducing into a somatic cell one or more transcription factors selected from the group consisting of OTX2, FOXG1, Pax6, Smad6, Lhx2, HNF4a, PKNOX2, Sox9, FoxDl, MITF, Mu-200, Mir204, Mir211, Crx, Six3, lf4, C-Myc, Klf9, RARa, Pax2, Smad3, Sox4, Soxl 1, Otxl, Pitx2, Nr2fl, Nr2f2, Tfec and any combination thereof,

2. A method of generating an induced photoreceptor cell, comprising introducing into a somatic cell one or more transcription factors selected from the group consisting of Ascll, Brn2, Rax, Crx, Nrl, NeuroDl, Otx2, Mirl24, FoxGl, Pax6, Ars2, Sox2, Nr2e3, Ror- beta, Blimpl, CBP, p300, Τ β2, PIAS3, Nr2el, Nrldl, Vsx2, Six6, RBI, Mirl 82, Mirl 83, Pax7 and any combination thereof.

3. A method of generating an induced retinal progenitor cell, comprising introducing into a somatic cell one or more transcription factors selected from the group consisting of Otx2, Hesl, c-Myc, Six3, Pax6, Rax, Ascll, Crx, Sox2, Vsx2 and any combination thereof.

4. A method of generating an induced retinal pigment epithelium cell, comprising introducing into a somatic cell the transcription factor OTX2 plus a cyclic AMP agonist molecule.

5. A method of generating an induced retinal pigment epithelium cell, comprising introducing into a somatic cell the transcription factors OTX2 and Sox9.

6. A method of generating an induced retinal pigment epithelium cell, comprising introducing into a somatic cell the transcription factors OTX2 and MITF.

7. A method of generating an induced retinal pigment epithelium cell, comprising introducing into a somatic cell the transcription factors OTX2, MITF and Sox9.

8. The method of claim 7, further comprising introducing into the somatic cell the transcription factor FoxDl.

9. The method of claim 7 or 8, further comprising introducing into the somatic cell the transcription factor P NOX2.

10. The method of any of claims 7-9, further comprising introducing into the somatic cell the transcription factors Mir200, Mir204 and Mir211.

11. The methods of claim 7 or 8, further comprising introducing into the somatic cell the transcription factor c-myc.

12. The method of claim 7 or 8, further comprising introducing into the somatic cell the transcription factor Klf-4.

13. The method of any of claims 4-6, further comprising introducing into the somatic cell the transcription factor HNF4a.

14. The method of any of claims 4-6, further comprising introducing into the somatic cell one or more transcription factors selected from the group consisting of FOXGl, Smad6, Lhx2, HNF4a and any combination thereof.

15. The method of any of claims 4-8, further comprising introducing into the somatic cell one or more transcription factors selected from the group consisting of Klf4, c- myc, lf9, RARa, Pax2, Smad3, Sox4, Soxl 1, Otxl, Pitx2, Nr2fl , Nr2f2, Tfec and any combination thereof.

16. A method of generating an induced photoreceptor cell, comprising introducing into a somatic cell one or more transcription factors selected from the group consisting of Ascll, Crx, Nrl, NeuroDl, Rax, Otx2, Mirl24, FoxGl, Pax6, Ars2 and any combination thereof.

17. The method of claim 16, further comprising introducing into the somatic cell one or more transcription factors selected from the group consisting of Nr2e3, Ror-beta, Blimpl, Mir200, CBP, p300, TRp2, PIAS3, Nr2el, Nrldl, Vsx2, Six6, RBI, Mirl82,

Mir 183, Pax7 and any combination thereof.

18. A method of generating an induced retinal progenitor cell, comprising introducing into a somatic cell the transcription factors mir200₃ mir204 and mir211.

19. The method of claim 18, further comprising introducing into the somatic cell the transcription factor MITF.

20. The method of claim 19, further comprising introducing into the somatic cell the transcription factor Smad6.

21. The method of claim 20, further comprising introducing into the somatic cell the transcription factors HNF4alpha_> Sox9, PKNOX2, Pax6, FoxDl, FoxGl and Otx2.

22. The method of claim 21, further comprising introducing into the somatic cell the transcription factor Lhx2.

23. The method of any of claims 18-22, further comprising introducing into the somatic cell a transcription factor selected from the group consisting of Sox2, Ikzfl, Ascll and any combination thereof.

24. The method of any preceding claim, wherein the somatic cell is a fibroblast or a white blood cell.

25. The method of any preceding claim, wherein the introducing comprises genetically modifying the somatic cell with one or more nucleic acid molecules comprising a nucleotide sequence encoding said transcription factors.

26. A genetically modified somatic cell produced by the method of claim 25.

27. An induced retinal pigment epithelium cell produced by the method of any of claims 1 or 4-15.

An induced photoreceptor cell produced by the method of any of claims 2, 16

29. An induced retinal progenitor ceil produced by the method of any of claims 3 or 18-23.

30. A method of treating a disorder of the eye in a subject in need thereof, comprising delivering to the eye(s) of the subject an effective amount of the cell of any of claims 27, 28 or 29.

31. The method of claim 30, wherein the disorder is selected from the group consisting of age-related macular degeneration, inherited macular degeneration, cystoid macular edema, retinal detachment, vascular occlusion, photoreceptor cell degeneration, infection, vision loss and any combination thereof.

32. A method of reprogramming a cell in the eye of a subject into an induced retinal pigment epithelium cell, comprising introducing into the cell of the eye one or more transcription factors selected from the group consisting of OTX2, FOXG1, Pax6, Smad6, Lhx2, HNF4a, PKNOX2, Sox9, FoxDl , MITF, Mir200, Mir204_; Mir211, Klf4, c-myc, Klf9, RARa, Pax2, Smad3, Sox4, Soxl 1, Otxl, Pitx2, Nr2fl, Nr2f2, Tfec and any combination thereof.

33. A method of reprogramming a cell in the eye of a subject into an induced photoreceptor cell, comprising introducing into the cell of the eye one or more transcription factors selected from the group consisting of Ascll, Crx, Nrl, NeuroDl, Rax, Otx2, Mirl24, FoxGl, Pax6, Ars2, Nr2e3, Ror-beta, Blimpl, Mir200, CBP, p300, TRp2, PI AS 3, Nr2el, Nrldl, Vsx2, Six6, RBI, Mirl82, Mirl83, Pax7 and any combination thereof.

34. A method of reprogramming a cell in the eye of a subject into an induced retinal progenitor cell, comprising introducing into the cell of the eye one or more transcription factors selected from the group consisting of Otx2, Hesl, c-Myc, Six3, Pax6, Rax, Ascll, Crx, Sox2, Vsx2 and any combination thereof.

35. A method of reprogramming a cell in the eye of a subject into an induced retinal pigment epithelium cell, comprising introducing into the cell of the eye the transcription factors OTX2, Sox9, FoxDl and MITF.

36. The method of claim 35, further comprising introducing into the cell the transcription factor PKNOX2,

37. The method of claim 35 or 36, further comprising introducing into the cell the transcription factors Mir200, Mir204 and Mir211.

38. The method of any of claims 35-37, further comprising introducing into the cell one or more transcription factors selected from the group consisting of FOXG1, Smad6, Lhx2, HNF4a and any combination thereof.

39. The method of any of claims 35-38, further comprising introducing into the cell one or more transcription factors selected from the group consisting of Klf4, c-myc, Klf9, RARa, Pax2, Smad3, Sox4, Soxl 1 , Otxl, Pitx2, Nr2fl, Nr2f2, Tfec and any combination thereof.

40. A method of reprogramming a cell in the eye of a subject into an induced photoreceptor cell, comprising introducing into the cell of the eye one or more transcription factors selected from the group consisting of Ascll, Crx, Nrl, NeuroDl, Rax, Otx2, Mirl24, FoxGl , Pax6, Ars2 and any combination thereof.

41. The method of claim 40, further comprising introducing into the cell one or more transcription factors selected from the group consisting of Nr2e3, Ror-beta, Blimp 1, Mir200, CBP, p300, TRP2, PIAS3, Nr2el, Nrldl, Vsx2, Six6, RBI, Mir 182, Mirl83, Pax7 and any combination thereof.

42. A method of reprogramming a cell in the eye of a subject into an induced retinal progenitor cell, comprising introducing into the cell of the eye the transcription factors mir200, mir204 and mir211.

43. The method of claim 42, further comprising introducing into the cell the transcription factor MITF.

44. The method of claim 43, further comprising introducing into the cell the transcription factor Smad6.

45. The method of claim 44, further comprising introducing into the cell the transcription factors HNF4alpha_; Sox9_s PKNOX2, Pax6, FoxDl, FoxGl and Otx2.

46. The method of claim 45, further comprising introducing into the cell the transcription factor Lhx2.

47. The method of any of claims 42-46, further comprising introducing into the cell a transcription factor selected from the group consisting of Sox2, Ikzfl, Ascll and any combination thereof.

48. The method of any of claims 32-47, wherein the cell in the eye is selected from the group consisting of a fibroblast, a retinal neuron, a Mueller glia cell and any combination thereof

49. The method of any of claims 32-48, wherein the subject is a human.

50. The method of any of claims 32-49, wherein the introducing comprises genetically modifying the cell of the eye with one or more nucleic acid molecules comprising a nucleotide sequence encoding said transcription factors.

51. The method of any of claims 32-50, wherein the subject has a disorder of the eye or is at risk of having a disorder of the eye.

52. The method of claim 51 , wherein the disorder is selected from the group consisting of age-related macular degeneration, inherited macular degeneration, cystoid macular edema, retinal detachment, vascular occlusion, photoreceptor cell degeneration, infection, vision loss and any combination thereof.

53. A method of reprogramming a preexisting retinal pigment epithelium (RPE) cell having no function or reduced/decreased/diminished function into a RPE cell having normal function or increased/improved/enhanced function, comprising introducing into the preexisting RPE cell one or more transcription factors selected from the group consisting of OTX2, FOXG1, Pax6, Smad6, Lhx2, HNF4a, PKNOX2, Sox9, FoxDl, MITF, Mir200, Mir204, Mir211, lf4, c-myc₅ Klf9, RARa, Pax2, Smad3, Sox4, Soxl 1, Otxl, Pitx2, Nr2fl, Nr2f2, Tfec and any combination thereof.