Cell therapies for retinal diseases

Abstract

The disclosure relates in some aspects to mammalian retinal pigment epithelial cells comprising an engineered activatable polynucleotide encoding a master regulatory transcription factor, compositions comprising the cells, methods of producing the cells, and methods of treatment using the cells. The disclosure also relates in some aspects to retinal grafts comprising induced pluripotent stem cell (iPSC)-derived retinal pigment epithelial cells, a proportion of which comprise an engineered polynucleotide comprising an inducible promoter operably linked to a neurogenin-2 coding sequence. Methods of using the retinal pigment epithelial cells and grafts for treating eye diseases are also provided herein.

Description

[0001] CELL THERAPIES FOR RETINAL DISEASES

[0002] RELATED APPLICATION

[0003] This application claims the benefit under 35 U.S.C. § 119(e) of U.S. provisional application number 63 / 594,181, filed on October 30, 2023, which is incorporated by reference herein in its entirety.

[0004] REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0005] The contents of the electronic sequence listing (J022770147WO00-SEQ-HJD.xml; Size: 13,446 bytes; and Date of Creation: October 25, 2024) is herein incorporated by reference in its entirety.

[0006] BACKGROUND

[0007] Retinal diseases, such as age-related macular degeneration, are the most common cause of blindness in the aging population, with an estimated 200 million cases worldwide. There are no effective treatments for the most prevalent (dry) form of the disease. Cell therapies, based on replacement of the retinal pigment epithelium with grafts made from human induced pluripotent stem cells are in early-stage trials around the world. These early-stage trials are designed mainly to assess the safety of the treatment. Thus far, the cell therapies are safe, and the grafted cells persist for long periods in patients. There are some preliminary indications that the treatment is providing some degree of benefit. While these safety trials are focused on patients whose disease is well advanced, trials are now beginning in patients with earlier stage disease, and these trials will provide a better indication of how effective the grafts are in arresting disease progression or improving vision.

[0008] SUMMARY

[0009] The present disclosure relates, in some aspects, to next-generation cell therapies for retinal diseases, such as age-related macular degeneration. Current approaches to retinal pigment epithelial cell (RPE) transplantation do not attempt to replace photoreceptors, and do not attempt to regenerate photoreceptors. Current approaches to photoreceptor replacement do not utilize retinal pigment epithelial cells derived from pluripotent stem cells, have not identified the differentiation state of the target retinal pigment epithelial cells required for efficient reprogramming from retinal pigment epithelial cells to photoreceptors, and do not generate photoreceptors and retinal pigment epithelium organized into appropriate retina-like anatomical structures. The present disclosure shows that retinal pigment epithelial cells generated from pluripotent stem cells (PSCs) comprising an engineered activatable polynucleotide encoding a master regulatory transcription factor (e.g., a polynucleotide encoding an inducible promoter operably linked to a master regulatory transcription factor) can generate photoreceptors. Further, the results of the studies described herein identify an optimal RPE cell differentiation state (e.g., age) for efficient reprogramming into photoreceptors. Surprisingly, the mixed population of RPE cells described herein (e.g., naturally occurring / unmodified RPE cells and engineered RPE cells expressing an activatable polynucleotide expressing NGN2) are capable of forming a bilayer resembling the natural state of the retina.

[0010] Some aspects of the present disclosure relate to a retinal graft comprising a pluripotent stem cell (PSC)-derived retinal pigment epithelial cells, a proportion of which comprise an engineered polynucleotide comprising an inducible promoter operably linked to a neurogenin-2 coding sequence. In some embodiments, the PSC is an induced PSC (iPSC). In other embodiments, the PSC is an embryonic stem cell.

[0011] In some embodiments, the retinal graft comprises at least one substrate selected from poly(lactic-co-glycolic acid, polycaprolactone, collagen, fibrin, hyaluronic acid, decellularized matrices, hydrogels.

[0012] In some embodiments, the PSC-derived retinal pigment epithelial cells are human cells. In some embodiments, the PSC-derived retinal pigment epithelial cells have been cultured for no more than 10-14 days. In some embodiments, the PSC-derived retinal pigment epithelial cells express neural cell adhesion molecule 1 (NCAM1). In some embodiments, the PSC-derived retinal pigment epithelial cells express melanocyte inducing transcription factor (MITF) and tight junction protein 1 (ZO1).

[0013] In some embodiments, the proportion of iPSC-derived retinal pigment epithelial cells comprising the engineered polynucleotide comprising an inducible promoter operably linked to a neurogenin-2 coding sequence is about 30% to about 70%, optionally about 50%.

[0014] In some embodiments, the inducible promoter is a chemical-inducible promoter. In some embodiments, the chemical-inducible promoter is a tetracycline-inducible promoter.

[0015] Further aspects of the disclosure relate to methods of treating an eye disorder in a subject, the method comprising administering the retinal graft of the present disclosure to an eye of a subject in need thereof. In some embodiments, the method comprises administering to the subject an inducing agent that activates the inducible promoter. In some embodiments, the inducible promoter is a tetracycline-inducible promoter, and the inducing agent is doxycycline. In some embodiments, an inducing agent is administered to the subject on the same day the retinal graft is administered (e.g., when the RPE cells are about 10 to about 14 days into differentiation from PSCs).

[0016] In some embodiments, the eye disorder is characterized by loss of photoreceptors. In some embodiments, the eye disorder is age-related macular degeneration.

[0017] Some aspects of the present disclosure relate to methods of producing a retinal graft comprising delivering, to pluripotent stem cells (PSCs) (e.g., embryonic stem cells or induced pluripotent stem cells), a polynucleotide comprising an inducible promoter operably linked to a neurogenin-2 coding sequence; and culturing the PSCs in differentiation media for about 10-14 days to produce the PSC-derived retinal pigment epithelial cells of the retinal graft the present disclosure. In some embodiments, the method further comprises delivering to the retinal graft an inducing agent, for example, shortly prior to (e.g., less than 24 hours prior to) delivering the retinal graft to a subject. In some embodiments, the method further comprises delivering to the retinal graft an inducing agent about 18 hours, about 12 hours, about 6 hours, about 3 hours, about 2 hours about 1 hours, or about 30 minutes prior to delivering the retinal graft to a subject. In some embodiments, the method further comprises delivering to the retinal graft an inducing agent within 30 minutes, within 20 minutes within 10 minutes, or within 5 minutes prior to delivering the retinal graft to a subject.

[0018] In some embodiments, the method further comprises producing a retinal graft by combining the PSC-derived retinal pigment epithelial cells with at least one substrate selected from poly(lactic-co-glycolic acid, polycaprolactone, collagen, fibrin, hyaluronic acid, decellularized matrices, hydrogels.

[0019] In some embodiments, the method further comprises administering the retinal graft to an eye of a subject having an eye disorder characterized by loss of photoreceptors.

[0020] In some embodiments, the eye disorder is characterized by loss of photoreceptors.

[0021] In some embodiments, the method further comprises administering to the subject an inducing agent that activates the inducible promoter.

[0022] In some embodiments, the inducible promoter is a tetracycline-inducible promoter, and the inducing agent is doxycycline. Some aspects of the present disclosure relate to a retinal pigment epithelial (RPE) cell comprising a polynucleotide having an inducible promoter operably linked to an open reading frame encoding a master regulatory transcription factor.

[0023] In some embodiments, the master regulatory transcription factor is selected from the group consisting of neurogenin 1 (NGN1), neurogenin 2 (NGN2), neurogenin 3 (NGN3), paired box 6 (PAX6), neuronal differentiation 1 (NEURODI), SIX homeobox 2 (SIX2), and orthodenticle homeobox 2 (0TX2). In some embodiments, the master regulatory transcription factor is NGN2.

[0024] In some embodiments, the RPE cell is differentiated from a pluripotent stem cell. In some embodiments, the pluripotent stem cell is a mammalian cell. In some embodiments, the pluripotent stem cell is a human cell. In some embodiments, the pluripotent stem cell is a human induced pluripotent stem cell. In some embodiments, the pluripotent stem cell is a human embryonic stem cell.

[0025] In some embodiments, the RPE cell (comprising the inducible promoter operably linked to an open reading frame encoding a master regulatory transcription factor) expresses one or more markers selected from the group consisting of neural cell adhesion molecule 1 (NCAM1), melanocyte inducing transcription factor (MITF), and / or tight junction protein 1 (ZO1). In some embodiments, the RPE cell expresses NCAM1. In some embodiments, RPE cells in a retinal graft that are not engineered, or otherwise do not express inducible promoter operably linked to an open reading frame encoding a master regulatory transcription factor, express markers of RPE maturation, such as folate receptor alpha (FOLR1), solute carrier family 16 member 8 (SLC16A8), ezrin, BEST1 and / or RPE65.

[0026] In some embodiments, the inducible promoter is a tetracycline-inducible promoter, and the inducing agent is doxycycline.

[0027] Some aspects of the present disclosure relate to a composition comprising the RPE cell of the present disclosure.

[0028] Some aspects of the present disclosure relate to a method of treating a retinal disease comprising administering to a subject having a retinal disease the RPE cell of the present disclosure and an inducing agent.

[0029] Some aspects of the present disclosure relate to a bilayer culture comprising, a first population of retinal pigment epithelial (RPE) cells and a second population of RPE cells, wherein the first population of the RPE cells comprises wild-type RPE cells and the second population of RPE cells comprises a polynucleotide having an inducible promoter operably linked to an open reading frame encoding a master regulatory transcription factor.

[0030] In some embodiments, the first population of RPE cells and the second population of RPE cells are in a ratio of 50:50.

[0031] Some aspects of the present disclosure relate to a method of treating a retinal disease comprising, administering to a subject having a retinal disease the bilayer culture of the present disclosure and a substrate suitable for transplantation into an eye.

[0032] In some embodiments, the method further comprises administering an inducing agent. In some embodiments, the inducing agent is doxycycline.

[0033] Some aspects of the present disclosure relate to a method of differentiating a retinal pigment epithelial (RPE) cell comprising, delivering to a pluripotent stem cell a polynucleotide having an inducible promoter operably linked to an open reading frame encoding a master regulatory transcription factor; and culturing the pluripotent stem cell to produce the RPE cell.

[0034] In some embodiments, the pluripotent stem cell is cultured for 10-70 days. In some embodiments, the pluripotent stem cell is cultured in a three-dimensional culture. In some embodiments, the three-dimensional culture comprises a scaffold, to produce a tissue sheet, spheroid, or an organoid.

[0035] In some embodiments, the RPE cell is purified by flow cytometry or magnetic bead sorting.

[0036] BRIEF DESCRIPTION OF THE DRAWINGS

[0037] FIG. 1 is a schematic showing a protocol for differentiation of human induced pluripotent stem cells (hiPSCs) into retinal pigment epithelial (RPE) cells. RPE cultures are established on day 14 and Neurogenin-2 (NGN2) induction begins on day 12-14, continuing up to Day 21.

[0038] FIGs. 2A-2C show immunofluorescence microscopy of markers of RPE, photoreceptor, and early eye cell populations. FIG. 2A shows immunostaining of the RPE marker Microphtalmia-associated transcription factor (MITF) and the photoreceptor markers Rhodopsin (RHO), the homeobox-containing transcription factor CHX10, and the homeobox transcription factor CRX in cultured control RPE cells (left column) or RPE cells in which NGN2 was induced (right column). FIG. 2B shows immuno staining of the RPE markers Bestrophin-1 (BEST1), Retinoid isomerohydrolase (RPE65), the early eye lineage marker Orthodenticle homeobox 2 (OTX2), and the photoreceptor marker Recoverin (RCVRN) in cultured control RPE cells (left column) or RPE cells in which NGN2 expression was induced (right column). FIG. 2C shows immuno staining of the early eye lineage marker Paired box 6 (PAX6), RPE marker Serpin family F member 1 (SERPINFl)and the photoreceptor marker Microtubule associated protein 2 (MAP2) in cultured control RPE cells (left column) or RPE cells in which NGN2 expression was induced (right column).

[0039] FIG. 3 are phase contrast images showing pluripotent stem cells (PSC) having a polynucleotide encoding an inducible promoter and the master regulatory transcription factor, NGN2, on Day 0 of the IPSC Differentiation Protocol described in Example 1, with the induction of expression of NGN2 in PSC on Day 0, which yielded cortical neurons. Phase contrast images of induced cells are shown at the time points indicated (Days 10, 11, and 14).

[0040] FIGs. 4A-4B show induction of NGN2 in hiPSC-derived mature RPE. Cells in Passage 2 of maturation phase were exposed to DOX for increasing periods. At 96 hours, there is no induction of photoreceptor progenitor cells as measured by TUBB3 staining (the few cells shown are neurons that sometimes persist as contaminants in RPE preparations; these cells are also present in uninduced controls). Photoreceptor progenitor cells appear (based on TUBB3 staining) after only 42 days of exposure to Doxycline. This contrasts with immature RPE, which shows induction of typical TUBB3 photoreceptor staining within 96 hours of DOX addition.

[0041] FIGs. 5A-5E are immunofluorescent micrographs showing marker expression during RPE differentiation using the IPSC Differentiation Protocol described in Example 1. Indirect Immunofluorescence micrographs of cultures at Days 1, 2, 4, 6, 8, 10, and 12 are shown in FIGs. 5A-5D. The markers tested include ZO1 and OTX2 (FIG. 5A), PAX6 and MITF (FIG. 5B), NCAM (FIG. 5C), and KI67 (FIG. 5D). Indirect Immunofluorescence micrographs of markers ZO1 and OTX2 at Day 12 are shown in FIG. 5E.

[0042] FIG. 6 are phase contrast images showing control and NGN2 RPE at 72 hours (hrs) postinduction. NGN2 RPE cells are RPE cells that have a polynucleotide encoding an inducible promoter and the master regulatory transcription factor, NGN2, which have been induced and undergone aggregation, as shown in FIG. 6. Small cells with photoreceptor-like morphology are present, and no RPE cells remain at 72 hrs post-induction as shown in FIG.6.

[0043] FIG. 7 is a schematic showing the desired orientation of RPE and photoreceptors following induction of a master regulatory transcription factor for grafts.

[0044] FIGs. 8A-8B are fluorescent images of a 50:50 mixture of wild type (WT) and Inducible NGN2 RPE in a monolayer. WT and NGN2 cells were plated in equal proportions and then induced with doxycycline (DOX). WT RPE and induced RPE cells were found in discrete patches in monolayer. The following markers were tested in FIG. 8A, DAPI, MAP2, TUBB3, and VSNL1. The following markers were tested in FIG. 8B, DAPI, BEST1, TUBB3, CRX. RPE cells were stained with DAPI.

[0045] FIGs. 9A-9B are confocal images showing a bilayer culture of a monolayer of RPE, which expressed markers of mature RPE along with large foci of cells that expressed markers of neural retina above the plane of the RPE monolayer. Cultures stained with BEST1, CRX, TUBB3, and VSNL are shown in FIG. 9A. BEST1 cells are basal, with photoreceptor-like cells arranged in clusters above the BEST1 layer. The clusters of photoreceptor- like cells were visible in the projection on the edges of the images in FIG. 9A. Rotated views of confocal projections of preparations stained with VSNL, TUBB3, BEST1, and CRX are shown in FIG. 9B. Photoreceptor-like cells were seen in clusters above the plane of RPE cells (BEST1+) in monolayer in FIGs. 9A and 9B.

[0046] DETAILED DESCRIPTION

[0047] There are inherent limitations to the cell therapy approaches based on replacement of the retinal pigment epithelium (RPE) with grafts made from human induced pluripotent stem cells (hiPSCs) or embryonic stem cells. The RPE has a critical role in supporting light sensing cells, referred to as photoreceptors, and the loss of this support leads to photoreceptor death and loss of vision. Current therapies have no means for replacing lost photoreceptors, and by the time most patients are diagnosed, this photoreceptor loss has already progressed. Ultimately, direct replacement of the light sensing cells, along with replenishment of the RPE, will likely be required to reverse much of the deterioration in vision in patients with retinal diseases, such as age-related macular degeneration (AMD).

[0048] The disclosure, in some aspects, relates to the production of RPE cells having regenerative capacity by artificially activating one or more master regulatory switches that control how immature cells develop into fully mature cells. Without being bound by theory, turning on one or more of these switches should ‘reboot’ the RPE cells back to a developmental stage during which they can produce photoreceptors, or cause them to transdifferentiate into photoreceptors, for example.

[0049] Retinal Pigment Epithelial Cells

[0050] The retinal pigment epithelium (RPE) is an important monolayer of pigmented cells situated between the neural retina and the choroid in the eye. This pigmentation comes from melanin, which absorbs stray light and therefore prevents light scatter within the eye, enhancing the clarity of visual images. The RPE plays several roles in maintaining visual function and overall health of the retina. For example, it transports nutrients from the choroid (a vascular layer behind the RPE) to the photoreceptors (the light-sensitive cells in the retina). It also helps in transporting waste products from the photoreceptors to the choroid for removal. The RPE also plays a role in the visual cycle, a process that regenerates visual pigments in photoreceptor cells after they have been bleached by light.

[0051] As described herein, the term “differentiated” cell includes a cell that has undergone a process (differentiation) through which develops specific structures and functions characteristic of its specialized type. As described herein, the term “undifferentiated” cell includes a cell with the potential to develop (e.g., differentiate) into various types of specialized cells, for example, a stem cell. During embryonic development, the eye originates from the neural tube. A portion of the neural tube evaginates and forms the optic vesicle. The cells of the outer layer of the optic vesicle will give rise to the RPE, while the inner layer forms the neural retina. Initially, the optic vesicle comprises undifferentiated neuroepithelial cells. As development progresses, the cells of the outer layer differentiate into RPE cells. These cells become pigmented, which is a hallmark feature of RPE cells. This pigmentation arises from melanin and is essential for absorbing stray light, preventing light scatter, and ensuring that only light which passes through the photoreceptors reaches the RPE. Various molecular biomarkers can be used to distinguish eye progenitors, early RPE cells, mature RPE cells, and neural retinal cells, such as photoreceptors. For example: eye progenitor cells may express one or more biomarkers selected from retina and anterior neural fold homeobox (RAX), paired box 6 (PAX6), visual system homeobox 2 (VSX2, CHX10), SIX homeobox 3 (SIX3), neural cell adhesion molecule 1 (NCAM1), and orthodenticle homeobox 2 (0TX2); early RPE cells may express one or more biomarkers selected from melanocyte inducing transcription factor (MITF), serpin family F member 1 (SERPINF1), and premelanosome protein (PMEL); mature RPE cells may express one or more biomarkers selected from retinoid isomerohydrolase RPE65 (RPE65), bestrophin 1 (BEST1), folate receptor alpha (FOLR1), solute carrier family 16 member 8 (SLC16A8), and neural retinal cells may express one or more biomarkers selected from cone-rod homeobox (CRX), CHX10, S-OPSIN, 0PN1SW, rhodopsin (RHO), POU class 4 homeobox 2 (POU4F2 / BRN3), recoverin (RCVRN), and microtubule associated protein 2 (MAP2). Cell surface markers, such as NCAM1, FOLR1, SLC16A8, PDGFRA, or any combination thereof, may be used to purify populations of eye progenitors or mature RPE cells, for example, using flow cytometry or magnetic cell sorting. In some embodiments, purification of RPE cell is performed using flow cytometry. In some embodiments, purification of RPE cell is performed using magnetic cell sorting. In some embodiments, purification of RPE cell is performed using magnetic bead sorting.

[0052] In some embodiments, an eye progenitor cell expresses at least 1 (e.g., at least 1, at least

[0053] 2, at least 3, at least 4, at least 5, at least 6) biomarkers selected from RAX, PAX6, CHX10, SIX3, NCAM1, and 0TX2. In some embodiments, an eye progenitor cell expresses at least 1, at least 2, at least 3, at least 4, at least 5, or at least 6 biomarkers selected from RAX, PAX6, CHX10, SIX3, NCAM1, and 0TX2. In some embodiments, an eye progenitor cell expresses RAX. In some embodiments, an eye progenitor cell expresses PAX6. In some embodiments, an eye progenitor cell expresses CHX10. In some embodiments, an eye progenitor cell expresses SIX3. In some embodiments, an eye progenitor cell expresses NCAM1. In some embodiments, an eye progenitor cell expresses 0TX2.

[0054] In some embodiments, an early RPE cell expresses at least 1 (e.g., at least 1, at least 2, at least 3) biomarkers selected from MITF, SERPINF1, ZO1 and PMEL. In some embodiments, an early RPE cell expresses at least 1, at least 2, or at least 3 biomarkers selected from MITF, SERPINF1, ZO1, and PMEL. In some embodiments, an early RPE cell expresses MITF. In some embodiments, an early RPE cell expresses SERPINF1. In some embodiments, an early RPE cell expresses PMEL.

[0055] In some embodiments, a mature RPE cell expresses at least 1 (e.g., at least 1, at least 2, at least 3, at least 4, at least 5) biomarkers selected from RPE65, BEST1, FOLR1, SLC16A8, and EZRIN (EZR). In some embodiments, a mature RPE cell expresses at least 1, at least 2, at least

[0056] 3, at least 4, at least 5 biomarkers selected from RPE65, BEST1, FOLR1, SLC16A8, and EZRIN (EZR). In some embodiments, a mature RPE cell expresses RPE65. In some embodiments, a mature RPE cell expresses BEST1. In some embodiments, a mature RPE cell expresses FOLR1. In some embodiments, a mature RPE cell expresses SLC16A8. In some embodiments, a mature RPE cell expresses EZRIN (EZR).

[0057] In some embodiments, a neural retinal cell expresses at least 1 (e.g., at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7) biomarkers selected from CRX, CHX10, S- OPSIN, RHODOPSIN, BRN3, RCVRN, and MAP2. In some embodiments, a neural retinal cell expresses at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, or at least 7 biomarkers selected from CRX, CHX10, S-OPSIN, RHODOPSIN, BRN3, RCVRN, and MAP2. In some embodiments, a neural retinal cell expresses CRX. In some embodiments, a neural retinal cell expresses CHX10. In some embodiments, a neural retinal cell expresses S-OPSIN. In some embodiments, a neural retinal cell expresses RHODOPSIN. In some embodiments, a neural retinal cell expresses BRN3. In some embodiments, a neural retinal cell expresses RCVRN. In some embodiments, a neural retinal cell expresses MAP2.

[0058] In some embodiments, the RPE cell expresses one or more (e.g., one or more, two or more, three or more, four or more, five or more) markers selected from NCAM1, FOLR1, SLC16A8, EZR, MITF, tight junction protein (ZO1; NCBI Gene ID: 7082), or any combination thereof. In some embodiments, the RPE cell expresses one or more, two or more, three or more, four or more, five or more markers selected from NCAM1, FOLR1, SLC16A8, EZR, MITF, ZO1, or any combination thereof. In some embodiments, the RPE cell expresses NCAM1. In some embodiments, the RPE cell is positive for NCAM1. In some embodiments, the RPE cell does not express or express low levels of markers of proliferating cells. Non-limiting examples of markers of proliferating cells include KI67. In some embodiments, the RPE expresses NCAM1 and does not express KI67. In some embodiments, the RPE expresses NCAM1 and expresses low levels of KI67.

[0059] Retinal pigment epithelial cells of the disclosure, in some embodiments, comprise an engineered (not naturally occurring) activatable polynucleotide encoding a master regulatory transcription factor. A polynucleotide is considered activatable if, for example, transcription of that polynucleotide can be turned on (activated), in vivo or ex vivo, in response to specific stimuli or conditions (e.g., inducing agent) or in response to the removal of specific stimuli or conditions (e.g., inducing agent). Likewise, transcription of an activatable polynucleotide can be turned off (deactivated) in response to specific stimuli or conditions (e.g., inducing agent) or in response to the removal of specific stimuli or conditions (e.g., inducing agent).

[0060] In some embodiments, an activatable polynucleotide comprises an inducible promoter. A promoter is a deoxyribonucleic acid (DNA) sequence that controls the transcription of a downstream open reading frame to which it is operably linked. Inducible promoters can be turned on (and, in some embodiments, off) in response to specific stimuli or conditions in vivo or ex vivo. Inducible promoters can be regulated by molecules including, but not limited to, doxycycline; RNA polymerase (e.g., T7 RNA polymerase); an estrogen receptor; an estrogen receptor fusion.

[0061] Non-limiting examples of inducible promoter systems include the doxycycline (Dox) system, the tetracycline (Tet) system, the Lac system, the Gal system, the alcohol oxidase (AOX1) promoter, steroid hormone receptors, the ecdysone system, heat-shock promoters, and other chemical-induced promoters (e.g., tetracycline, estradiol, cumate, or mifepristone), or synZiFTR switches. In some embodiments, an inducible system is a doxycycline system, tetracycline system, Lac system, Gal system, alcohol oxidase promoter, steroid hormone receptors, ecdysone system, heat-shock promoters, tamoxifen-inducible system, or isopropyl P- D-l- thiogalactopyranoside (IPTG)-inducible system. In some embodiments, an inducible promoter is a chemical-inducible promoter. In some embodiments, a chemical-inducible promoter is a tetracycline-inducible promoter.

[0062] Some aspects of the disclosure contemplate activation of a master regulatory transcription factor in an RPE cell in vivo, for example, after the engineered RPE cells (e.g., eye progenitor cells or mature RPE cells) are transplanted into an eye of a subject, for example. For example, an inducing agent may be administered to a subject who has received a graft described herein to activate transcription of a transcription factor (e.g., NGN2).

[0063] Other genetic elements may be harnessed to regulate expression of an activatable polynucleotide. For example, hormone-responsive elements may be included upstream of an open reading frame encoding a master regulatory transcription factor. CRISPR / Cas-based systems may also be used, including CRISPRa (activation), which uses a deactivated Cas9 protein (dCas9) fused to one or more transcriptional activators. When targeted to specific gene promoters by guide RNAs, the fusion protein can activate gene transcription. An enhanced version of CRISPRa, referred to as synergistic activation mediator (SAM), where dCas9 is combined with multiple activation domains to boost transcriptional activation, may also be used. Other systems include, for example, RNA-based systems, such as riboswitches and synthetic small RNAs, and protein degradation systems that use, for example, specific degron sequences.

[0064] In some embodiments, the inducible promoter system of the present disclosure is a tetracycline-inducible system. The tetracycline-inducible system comprises two complementary control circuits, initially described as the tTA dependent (Gossen et al. Proc Natl Acad Sci USA. 1992 Jun 15 ; 89(12): 5547-51 ) and rtTA dependent (Gossen et al. Science. 1995 Jun 23;268(5218): 1766-9) expression systems, commonly referred to as the Tet-Off system (tTA dependent) and the Tet-On system (rtTA dependent). In the Tet-Off system and the Tet-On system, a recombinant tetracycline controlled transcription factor (tTA or rtTA) interacts with a tTA / rtTA responsive promoter, Ptet, to drive expression of the gene of interest. Expression is regulated by the effector substance tetracycline (Tc) or one of its derivatives. Tet-On systems respond to doxycycline (Dox). Tetracyclines act at the level of DNA binding of tetracycline- controlled transactivator (tTA) and reverse tetracycline-controlled transactivator (rtTA) transcription factors. rtTA requires a tetracycline ligand for DNA binding and transcription. By contrast, the interaction between tTA and DNA is prevented by tetracycline. Thus, the two versions of the Tet system respond to tetracyclines differently and may be used in a complementary manner. In some embodiments, doxycycline, a tetracycline derivative, is the effector substance used for a Tet-On or a Tet-Off system. Doxycycline binds with high affinity to tTA as well as to rtTA and, thus, is fully effective in a Tet-Off system at concentrations as low as 1-2 ng / ml in the case of tTA, for example. In a Tet-On system, concentrations as low as 80 ng / ml, in the case of rtTA2-synl, for example, are effective.

[0065] In some embodiments, the inducible promoter system of the present disclosure is a IPTG- Inducible System. In some embodiments, the IPTG-Inducible System comprises the presence of a lactose (lac) repressor protein and a lac operon. The lac repressor is a DNA-binding protein that binds to the lac operon and inhibits expression of a nucleic acid operably linked to the lac operon. The presence of allolactose or an allolactose mimic, such as isopropyl 0-0-1- thiogalactopyranoside (IPTG), inhibits the DNA binding ability of the lac repressor protein. This loss of DNA binding by the lac repressor is used for transcriptional activation of the lac operon and expression of any nucleic acid linked to that operon. The lac operon contains three structural genes, and a promoter, a terminator, regulator, and an operator. The three structural genes are lacZ, lacY, and lac A. lacZ encodes 0-galactosidase (LacZ), an intracellular enzyme that cleaves the disaccharide lactose into glucose and galactose; lacY encodes lactose permease (LacY), a transmembrane symporter that pumps 0-galactosides into the cell using a proton gradient in the same direction; and lacA encodes galactoside O-acetyltransferase (LacA), an enzyme that transfers an acetyl group from acetyl-CoA to 0-galactosides.

[0066] In some embodiments, the inducible promoter system of the present disclosure is a Tamoxifen-Inducible System. In some embodiments, the Tamoxifen-Inducible System are engineered constructs comprising (a) a promoter operably linked to a nucleic acid encoding a master regulatory transcription factor, wherein the nucleic acid encoding the master regulatory transcription factor is flanked by estrogen receptor (ERT2) sequences, and (b) a deoxyribonucleic acid (DNA)-binding recognition sequence.

[0067] PSC-Derived RPE cells

[0068] The RPE cells of the disclosure, in some aspects, are produced from pluripotent stem cells (PSCs), for example, human PSCs or human embryonic stem (ES) cells. In some embodiments, the RPE cells of the present disclosure are differentiated from PSCs (e.g., iPSCs). In some embodiments, the RPE cells of the present disclosure are differentiated from PSCs (e.g., iPSCs)using cell culture protocols known in the art. For example, see Foltz, Leah P, and Dennis O Clegg. “Rapid, Directed Differentiation of Retinal Pigment Epithelial Cells from Human Embryonic or Induced Pluripotent Stem Cells.” Journal of visualized experiments : JoVE ,128 56274. 30 Oct. 2017, doi: 10.3791 / 56274. In some embodiments, the PSCs are induced PSCs (iPSCs), which include pluripotent stem cells derived from adult somatic cells that have been reprogrammed back into an embryonic-like state. This reprogramming allows the cells to develop into any type of cell in the body. In some embodiments, the PSCs are human induced PSCs (hiPSCs). Other non-limiting examples of PSCs from which RPE cells may be derived include embryonic stem cells, for example, human embryonic stem cells. Adult stem cells may also be used, in some embodiments.

[0069] In some embodiments, the pluripotent stem cell is an induced pluripotent stem cell (iPSC). In some embodiments, the pluripotent stem cell is a human induced pluripotent stem cell (hiPSC). In some embodiments, the pluripotent stem cell (e.g., iPSC, hiPSC) are differentiated to retinal pigment epithelial cells (RPE), referred to as “the iPSC-derived retinal pigment epithelial cells”. In some embodiments, the iPSC-derived retinal pigment epithelial cells are human cells. In some embodiments, the iPSC-derived retinal pigment epithelial cells express neural cell adhesion molecule 1 (NCAM1). In some embodiments, the iPSC-derived retinal pigment epithelial cells express melanocyte inducing transcription factor (MITF) and tight junction protein 1 (ZO1).

[0070] Pluripotent stem cells (e.g., iPSCs or ES cells) may be cultured in media. In some embodiments, the pluripotent stem cells are in a differentiation phase. As described herein, the term “differentiation phase” refers to a process by which pluripotent stem cells (e.g., iPSCs) are differentiated into fully mature RPE cells. For example, see FIG. 1, which is an example of a cell culture protocol including a differentiation phase, from Day 0 to Day 14, using pluripotent stem cells (e.g., iPSC) cultured in retinal pigment epithelium differentiation media (RDM). Pluripotent stem cells (e.g., iPSCs) in the differentiation phase, which are not fully mature RPE cells may be described as an intermediate stage of differentiation. Examples of pluripotent stem cells (e.g., iPSCs) that are not fully mature include eye progenitors and early RPE cells.

[0071] Mature-Derived RPE Cells

[0072] Some aspects of the present disclosure relate to methods and compositions comprising mature-derived RPE cells. As described herein, the term “mature-derived RPE cells” refers to an RPE cell produced from the differentiation of pluripotent stem cells (e.g., iPSCs) to fully mature RPE cells. RPE cells in a fully mature state, for example, express specific proteins. Examples of proteins expressed in a fully mature RPE cell include but are not limited to, RPE65, BEST1, F0LR1, SLC16A8, and EZRIN (EZR). In some embodiments, the mature-derived RPE cell is a wild type RPE cell. Examples of mature-derived RPE cells are shown in the protocol in FIG. 1 starting on D14, labeled as “MATPO”, which is the beginning of the maturation phase. As described herein, the term “maturation phase” refers to a period of time where a cell that has been differentiated reaches a fully mature state and cell fate does not change. RPE cells that are early in the maturation phase refers to cells that have recently reached full maturity, for example, D14-D28 of the protocol in FIG. 1.

[0073] Master Regulatory Transcription Factors

[0074] One or more master regulatory transcription factors may be expressed in an RPE cell to change the cell fate of the RPE cell, for example. Master regulatory transcription factor proteins have an important role in determining cell identity and fate. In the context of development, differentiation, and cell reprogramming, these transcription factors can bind to specific DNA sequences and control the expression of downstream target genes, often acting as pivotal switches in cellular programs. When expressed in a certain cellular context, they can drive the entire cell towards a specific lineage or fate by orchestrating a cascade of gene expression changes. For example, it is well known that the master regulatory transcription factors octamer- binding protein 4 (0CT4; NCBI Gene ID: 5460), SRY-box transcription factor 2 (SOX2; NCBI Gene ID: 6657), Nanog homeobox (NANOG; NCBI Gene ID: 79923), and lin-28 homolog A (LIN28; NCBI Gene ID: 79727) are essential for maintaining embryonic stem cell identity and pluripotency.

[0075] Non-limiting examples of master regulatory transcription factors in the eye include paired box 6 (PAX6; NCBI Gene ID: 5080), cone-rod homeobox (CRX; NCBI Gene ID: 1406), RAR related orphan receptor B (RORB; NCBI Gene ID: 6096), visual system homeobox 2 (VSX2, CHX10; NCBI Gene ID: 338917), SIX homeobox 3 (SIX3; NCBI Gene ID: 6496), SIX homeobox 6 (SIX6; NCBI Gene ID: 4990), LIM homeobox 2 (LHX2; NCBI Gene ID: 9355), melanocyte inducing transcription factor (MITF; NCBI Gene ID: 4286), SRY-box transcription factor 2 (SOX2; NCBI Gene ID: 6657), prospero homeobox 1 (PROXI; NCBI Gene ID: 5629), forkhead box Cl (FOXCI; NCBI Gene ID: 2296), paired like homeodomain 2 (PITX2; NCBI Gene ID: 5308), orthodenticle homeobox 2 (OTX2; NCBI Gene ID: 5015), and atonal bHLH transcription factor 7 (ATOH7 / MATH5; NCBI Gene ID: 220202). Non-limiting examples of master regulatory transcription factors in the nervous system include SOX2, neurogenin 1 (NGN1; NCBI Gene ID: 4762), neurogenin 2 (NGN2; NCBI Gene ID: 63973), neurogenin 3 (NGN3; NCBI Gene ID: 50674), achaete-scute family bHLH transcription factor 1 (ASCL1 / MASH1; NCBI Gene ID: 429), neuronal differentiation 1 (NEURODI; NCBI Gene ID: 4760), oligodendrocyte transcription factor 1 (OLIG1; NCBI Gene ID: 116448), oligodendrocyte transcription factor 2 (OLIG2; NCBI Gene ID: 10215), NK2 homeobox 2 (NKX2.2; NCBI Gene ID: 4821), NK6 homeobox 1 (NKX6.1; NCBI Gene ID: 4825), PAX6, T-box brain transcription factor 1 (TBR1; NCBI Gene ID: 10716), eomesodermin (TBR2; NCBI Gene ID: 8320), forkhead box G1 (FOXG1; NCBI Gene ID: 2290), empty spiracles homeobox 1 (EMX1; NCBI Gene ID: 2016), empty spiracles homeobox 2 (EMX2; NCBI Gene ID: 2018), distal-less homeobox (DLX) FAMILY (e.g., DLX1; NCBI Gene ID: 1745, DLX2; NCBI Gene ID: 1746), ATOH7 (MATH5), LIM homeobox transcription factor 1 alpha (LMX1A; NCBI Gene ID: 4009), and LIM homeobox transcription factor 1 beta (LMX1B; NCBI Gene ID: 4010). In some embodiments, an activatable polynucleotide encodes (e.g., comprises an open reading frame encoding) one or more (e.g., 1, 2, 3, or 4) master regulatory transcription factor(s) selected from NGN1, NGN2, NGN3, PAX6, NEURODI, SIX3, and OTX2. In some embodiments, an activatable polynucleotide encodes (e.g., comprises an open reading frame encoding) NGN1. In some embodiments, an activatable polynucleotide encodes (e.g., comprises an open reading frame encoding) NGN2. In some embodiments, an activatable polynucleotide encodes (e.g., comprises an open reading frame encoding) NGN3. In some embodiments, an activatable polynucleotide encodes (e.g., comprises an open reading frame encoding) PAX6. In some embodiments, an activatable polynucleotide encodes (e.g., comprises an open reading frame encoding) NEURODI. In some embodiments, an activatable polynucleotide encodes (e.g., comprises an open reading frame encoding) SIX3. In some embodiments, an activatable polynucleotide encodes (e.g., comprises an open reading frame encoding) OTX2.

[0076] In some embodiments, an activatable polynucleotide encodes (e.g., comprises an open reading frame encoding) NGN2. A non-limiting example of a human NGN2 polynucleotide sequence is provided in Gene ID: 63973. Non-limiting examples of cDNA or mRNA sequences of NGN2 are provided in NCBI accession number NM_024019.4. Non-limiting examples of a NGN2 protein sequence encoded by the polynucleotide of the present disclosure is provided in NCBI accession number NP_076924.1.

[0077] NGN2 amino acid sequence (NCBI accession number NP_076924.1; SEQ ID NO: 1) MFVKSETLELKEEEDVLVLLGSASPALAALTPLSSSADEEEEEEPGASGGARRQRGAEA GQGARGGVAAGAEGCRPARLLGLVHDCKRRPSRARAVSRGAKTAETVQRIKKTRRLK ANNRERNRMHNLNAALDALREVLPTFPEDAKLTKIETLRFAHNYIWALTETLRLADHC GGGGGGLPGALFSEAVLLSPGGASAALSSSGDSPSPASTWSCTNSPAPSSSVSSNSTSPYS CTLSPASPAGSDMDYWQPPPPDKHRYAPHLPIARDCI

[0078] In some embodiments, the polynucleotide comprises an open reading frame encoding a polypeptide comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the polynucleotide comprises an open reading frame encoding a polypeptide comprising an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the polynucleotide comprises an open reading frame encoding a polypeptide comprising an amino acid sequence having at least 85% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the polynucleotide comprises an open reading frame encoding a polypeptide comprising an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the polynucleotide comprises an open reading frame encoding a polypeptide comprising an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the polynucleotide comprises an open reading frame encoding a polypeptide comprising an amino acid sequence having at least 98% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the polynucleotide comprises an open reading frame encoding a polypeptide comprising an amino acid sequence having at least 99% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the polynucleotide comprises an open reading frame encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 1.

[0079] Retinal Grafts and Related Cell Populations

[0080] A population, or a subpopulation, of cells includes more than one cell of the same cell type or of different cell types (e.g., 2, 3, 4, 5 or more different cell types). The distinction between a population of cells and a subpopulation of cells is relative such that a population of cells includes a subpopulation of cells. That is, a “subpopulation” of cells is simply a collection of two or more cells, and in some embodiments, is part of a population of cells, which may be a larger population of cells. Herein, in some embodiments, reference is made to “a subpopulation of cells wherein each cell of the subpopulation is a mammalian retinal pigment epithelial cell”. This subpopulation of cells, in some embodiments, may be part of a greater population of cells that includes additional cell types, for example, retinal progenitor cells and / or retinal cell types selected from photoreceptor cells, retinal ganglion cells, and Muller glial cells.

[0081] In some embodiments, a population of cells, such as a population of retinal cell types, may be differentiated from a population or subpopulation of RPE cells, for example, RPE cells comprising an engineered activatable polynucleotide encoding a master regulatory transcription factor (e.g., NGN1, NGN2, NGN3, PAX6, NEURODI, SIX3, or 0TX2, preferably NGN2).

[0082] In some embodiments, a population of cells differentiated from RPE cells of the disclosure express one or more biomarkers selected from CRX, CHX10, S-OPSIN, RHODOPSIN (RHO), BRN3, RCVRN, MAP2, which are known photoreceptor lineage biomarkers. Thus, in some embodiments, a population of cells differentiated from RPE cells of the disclosure express one more photoreceptor lineage biomarkers. In some embodiments, such cells exhibit one or more functions that are characteristic of a photoreceptor. Photoreceptors are specialized neurons in the retina that transduce light into electrical signals, which then get processed by other cells in the retina, sent to the brain via the optic nerve, and interpreted as vision. Photoreceptors include rods, responsible for vision at low light levels, and cones, responsible for vision at higher light levels. Non-limiting examples of functional characteristics of photoreceptors include light sensitivity, color discrimination, resolution, response time, dark adaptation, photopigment regeneration, signal transduction, and synaptic transmission. In some embodiments cells of a population differentiated from RPE cells express CRX (i.e., are CRX+). In some embodiments cells of a population differentiated from RPE cells of the disclosure express RHO (i.e., are RHO+). In some embodiments cells of a population differentiated from RPE cells of the disclosure express CHX10 (i.e., are CHX10+). In some embodiments cells of a population differentiated from RPE cells of the disclosure express RCVRN (i.e., are RCVRN+). In some embodiments cells of a population differentiated from RPE cells of the disclosure express MAP2 (i.e., are MAP2+).

[0083] In other embodiments, a population of cells differentiated from RPE cells of the disclosure express one or more of RAX, PAX6, CHX10, SIX3, NCAM1, and 0TX2, which are early eye lineage biomarkers.

[0084] The number of cells in any particular population or subpopulation of cells can vary based on several factors, including, for example, the purpose and cell type; and, if being administered to a subject, the number of cells may depend on the location of delivery, route of administration, and / or delivery system. For example, the number of cells in a population or subpopulation of cells (e.g., use in a cell graft) may range from a few hundred to a few million cells. In some embodiments, the number of cells in a population or subpopulation of cells is about 100 to about 1000, about 100 to about 10,000, about 100 to about 100,000, about 1000 to about 10,000, about 1000 to about 100,000, about 10,000 to about 100,000, about 10,000 to about 1,000,000, or about 100,000 to about 1,000,000 cells. In some embodiments, the number of cells in a population or subpopulation of cells is about 50,000 to about 100,000 cells, or about 50,000 to about 200,000 cells, for example, in a cell suspension. In some embodiments, such as those in which the cells are organized into a sheet or monolayer on a scaffold or substrate, the number of cells may include about 1,000 to about 10,000, about 2,000 to about 8,000, or about 4,000 to about 6,000 cells per square millimeter. In some embodiments, the number of cells in a population or subpopulation of cells is about IxlO6to about 5xl06.

[0085] Also provided herein are compositions comprising a population or subpopulation of cells of the disclosure, such as RPE cells comprising an engineered activatable polynucleotide expressing a master regulatory transcription factor (e.g., NGN1, NGN2, NGN3, PAX6, NEURODI, SIX3, or OTX2, preferably NGN2). Compositions may include cells and other materials, for example an ocular graft or implant material. Non-limiting examples of such materials include hydrogels, bioengineered collagen matrices, polytetrafluoroethylene (PTFE), human acellular dermal matrix, and biocompatible metals and polymers such as titanium, polyimide, and silicone rubber. Other examples, including those for RPE grafts, include polyester, silk fibroin, and parylene. Compositions may also, or alternatively, include other reagents, such as enzymes, buffers and solutions, inhibitors, serum (e.g., human serum), culture media, or other nutrient-rich media or non-nutrient media. In some embodiments, a population of cells or a subpopulation of cells is cultured in cell culture media, thus, composition of the disclosure may include cell culture media.

[0086] In some embodiments, the present disclosure relates to a retinal graft. As described herein, the term “retinal graft” includes a product and / or a procedure to treat retinal disease by replacing retinal pigment epithelial cells and photoreceptor cells in the retina. In some embodiments, the retinal graft involves culturing RPE cells. In some embodiments, the RPE cells are differentiated from pluripotent stem cells (e.g., iPSC). In some embodiments, the RPE cells are harvested from a donor. In some embodiment, the pluripotent stem cells are derived from donor cells. As described herein, a “donor” includes a healthy donor. For example, a cell or cells may be isolated from a biological sample obtained from a donor, such as a healthy donor. As used herein “healthy donor” refers to a subject that does not have, or is not suspected of having, a retinal disease. However, in some embodiments, a donor cell is derived from a subject having (or suspected of having) a retinal disease, for example in the context of autologous cell therapy.

[0087] In some embodiments, the retinal graft comprises (a) induced pluripotent stem cell (iPSC)-derived retinal pigment epithelial cells that comprise an engineered polynucleotide comprising an inducible promoter operably linked to a neurogenin-2 coding sequence, and (b) mature-derived retinal pigment epithelial cells. Some proportion of the cells in the retinal graft comprise iPSC-derived retinal pigment epithelial cells that include the engineered polynucleotide, while another proportion of the cells in the retinal graft comprise mature-derived retinal pigment epithelial cells. In some embodiments, the proportion of iPSC-derived RPE cells comprising the engineered polynucleotide is derived from PSCs that have been cultured in differentiation media for about 10 to 12 days (e.g., 10, 11, or 12 days). In some embodiments, the proportion of PSC-derived RPE cells comprising the engineered polynucleotide are derived from PSCs that have been cultured in differentiation media for about 10, 11, or 12 days. In some embodiments, the proportion of PSC-derived RPE cells comprising the engineered polynucleotide are derived from PSC that have been cultured in differentiation media for about 10 days. In some embodiments, the proportion of PSC-derived RPE cells comprising the engineered polynucleotide are derived from PSC that have been cultured in differentiation media for about 11 days. In some embodiments, the proportion of PSC-derived RPE cells comprising the engineered polynucleotide are derived from PSC that have been cultured in differentiation media for about 12 days.

[0088] In some embodiments, the proportion of PSC-derived RPE cells comprising the engineered polynucleotide are mature-derived RPE in an early maturation phase (e.g., D14-D28 of protocol shown in FIG.l). In some embodiments, the retinal graft is produced by combining the PSC-derived retinal pigment epithelial cells with mature-derived retinal pigment epithelial cells.

[0089] In some embodiments, the mature-derived retinal pigment epithelial cells have been cultured for at least 21 (e.g., at least 21, 25, 30, 40, 50, 60, 70, 80, 90, or 100) days. In some embodiments, the mature-derived retinal pigment epithelial cells have been cultured for at least 21, 25, 30, 40, 50, 60, 70, 80, 90, or 100 days. In some embodiments, the mature-derived retinal pigment epithelial cells have been cultured for at least 21 days. In some embodiments, the mature-derived retinal pigment epithelial cells have been cultured for at least 25 days. In some embodiments, the mature-derived retinal pigment epithelial cells have been cultured for at least 30 days. In some embodiments, the mature-derived retinal pigment epithelial cells have been cultured for at least 40 days. In some embodiments, the mature-derived retinal pigment epithelial cells have been cultured for at least 50 days. In some embodiments, the mature-derived retinal pigment epithelial cells have been cultured for at least 60 days. In some embodiments, the mature-derived retinal pigment epithelial cells have been cultured for at least 70 days. In some embodiments, the mature-derived retinal pigment epithelial cells have been cultured for at least 80 days. In some embodiments, the mature-derived retinal pigment epithelial cells have been cultured for at least 90 days. In some embodiments, the mature-derived retinal pigment epithelial cells have been cultured for at least 100 days.

[0090] In some embodiments, the retinal graft has been treated with an inducing agent that activates the inducible promoter. In some embodiments, the retinal graft is treated with an inducing agent that activates the inducible promoter before the retinal graft is administered to the subject.

[0091] In some embodiments, the retinal graft comprises at a substrate suitable for transplantation in a human eye. In some embodiments, the substrate is selected from poly(lactic- co-glycolic acid, polycaprolactone, collagen, fibrin, hyaluronic acid, decellularized matrices, and hydrogels. In some embodiments, the retinal graft comprises at least one substrate selected from poly(lactic-co-glycolic acid, polycaprolactone, collagen, fibrin, hyaluronic acid, decellularized matrices, hydrogels. In some embodiments, the substrate is poly(lactic-co-glycolic acid. In some embodiments, the substrate is polycaprolactone. In some embodiments, the substrate is collagen. In some embodiments, the substrate is fibrin. In some embodiments, the substrate is hyaluronic acid. In some embodiments, the substrate is decellularized matrices. In some embodiments, the substrate is and hydrogels.

[0092] Other examples of substrates suitable for transplantation into a human eye, include, for example, hydrogels, polyester, silk fibroin, and parylene. This attachment occurs, in some embodiments, by simply culturing the RPE cells in the presence of the substrate. Thus, in some embodiments, the scaffold using in a 3D culture is or includes a substrate suitable for transplantation into a human eye.

[0093] In some embodiments, the retinal graft comprises a first population of retinal pigment epithelial (RPE) cells and a second population of RPE cells. In some embodiments, the first population of the RPE cells comprises wild type mature-derived RPE cells. In some embodiments, the second population of the RPE cells comprises wild type mature-derived RPE cells. In some embodiments, the first population of the RPE cells comprises RPE cells comprising a polynucleotide having an inducible promoter operably linked to an open reading frame encoding a master regulatory transcription factor. In some embodiments, the second population of the RPE cells comprises RPE cells comprising a polynucleotide having an inducible promoter operably linked to an open reading frame encoding a master regulatory transcription factor. In some embodiments, the first population of the RPE cells comprises RPE cells and the second population of RPE cells comprises a polynucleotide having an inducible promoter operably linked to an open reading frame encoding a master regulatory transcription factor. In some embodiments, the second population of the RPE cells comprises RPE cells and the first population of RPE cells comprises a polynucleotide having an inducible promoter operably linked to an open reading frame encoding a master regulatory transcription factor.

[0094] In some embodiments, the first population of RPE cells and the second population of RPE cells are in a ratio of about 35:65, about 40:60, about 45:55, or about 50:50. In some embodiments, the first population of RPE cells and the second population of RPE cells are in a ratio of about 35:65, 40:60, 45:55, 50:50. In some embodiments, the first population of RPE cells and the second population of RPE cells are in a ratio of 50:50.

[0095] In some embodiments, the proportion of iPSC-derived retinal pigment epithelial cells comprising the iPSC-derived retinal pigment epithelial cells is about 30% to 70% (e.g., 30% to 70% , 35% to 70%, 40% to 70%, 45% to 70%, 50% to 70%, 55% to 70%, 60% to 70%, 65% to 70%). In some embodiments, the proportion of iPSC-derived retinal pigment epithelial cells comprising the iPSC-derived retinal pigment epithelial cells is about 30% to 70% , 35% to 70%, 40% to 70%, 45% to 70%, 50% to 70%, 55% to 70%, 60% to 70%, 65% to 70%. In some embodiments, the proportion of iPSC-derived retinal pigment epithelial cells comprising the iPSC-derived retinal pigment epithelial cells is about 30% to 70% . In some embodiments, the proportion of iPSC-derived retinal pigment epithelial cells comprising the iPSC-derived retinal pigment epithelial cells is about 35% to 70%. In some embodiments, the proportion of iPSC- derived retinal pigment epithelial cells comprising the iPSC-derived retinal pigment epithelial cells is about 40% to 70%. In some embodiments, the proportion of iPSC-derived retinal pigment epithelial cells comprising the iPSC-derived retinal pigment epithelial cells is about 45% to 70%. In some embodiments, the proportion of iPSC-derived retinal pigment epithelial cells comprising the iPSC-derived retinal pigment epithelial cells is about 50% to 70%. In some embodiments, the proportion of iPSC-derived retinal pigment epithelial cells comprising the iPSC-derived retinal pigment epithelial cells is about 55% to 70%. In some embodiments, the proportion of iPSC-derived retinal pigment epithelial cells comprising the iPSC-derived retinal pigment epithelial cells is about 60% to 70%. In some embodiments, the proportion of iPSC- derived retinal pigment epithelial cells comprising the iPSC-derived retinal pigment epithelial cells is about 65% to 70%. In some embodiments, the proportion of iPSC-derived retinal pigment epithelial cells comprising the iPSC-derived retinal pigment epithelial cells is about 30% to about 70%, about 50%. In some embodiments, the proportion of iPSC-derived retinal pigment epithelial cells comprising the iPSC-derived retinal pigment epithelial cells is about 30% to about 70%, optionally about 50%. In some embodiments, the proportion of iPSC-derived retinal pigment epithelial cells comprising the iPSC-derived retinal pigment epithelial cells is 30% to 70%.

[0096] In some embodiments, the proportion of iPSC-derived retinal pigment epithelial cells comprising the iPSC-derived retinal pigment epithelial cells is about 30% (e.g., 30% , 35%, 40%, 45%, 50%, 55%, 60%, 65%). In some embodiments, the proportion of iPSC-derived retinal pigment epithelial cells comprising the iPSC-derived retinal pigment epithelial cells is about 30% , about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65% . In some embodiments, the proportion of iPSC-derived retinal pigment epithelial cells comprising the iPSC-derived retinal pigment epithelial cells is about 30%. In some embodiments, the proportion of iPSC-derived retinal pigment epithelial cells comprising the iPSC-derived retinal pigment epithelial cells is about 35%. In some embodiments, the proportion of iPSC-derived retinal pigment epithelial cells comprising the iPSC-derived retinal pigment epithelial cells is about 40%. In some embodiments, the proportion of iPSC-derived retinal pigment epithelial cells comprising the iPSC-derived retinal pigment epithelial cells is about 45%. In some embodiments, the proportion of iPSC-derived retinal pigment epithelial cells comprising the iPSC-derived retinal pigment epithelial cells is about 50%. In some embodiments, the proportion of iPSC-derived retinal pigment epithelial cells comprising the iPSC-derived retinal pigment epithelial cells is about 55%. In some embodiments, the proportion of iPSC-derived retinal pigment epithelial cells comprising the iPSC-derived retinal pigment epithelial cells is about 60%. In some embodiments, the proportion of iPSC-derived retinal pigment epithelial cells comprising the iPSC-derived retinal pigment epithelial cells is about 65%. In some embodiments, the proportion of iPSC-derived retinal pigment epithelial cells comprising the iPSC-derived retinal pigment epithelial cells is 50%.

[0097] In some embodiments, a population of cells or a subpopulation of cells is cryopreserved. In some embodiments, the cry opreservation is performed on cells in either the differentiation phase or the maturation phase of a culturing protocol, for example see FIG. 1. Methods of cryopreservation are well known in the art. Cells of the disclosure, in some embodiments, form tissue sheets, spheroids, or organoids, in vitro or in vivo (or both). Assembloids, which include a combination of organoids (e.g., a combination of different organoids), are also contemplated herein. A tissue sheet includes a layer of cells that has been grown in vitro (outside the body) and is typically intended for transplantation into the eye. It may include a thin, planar structure of cell aggregates, similar to a sheet of paper or fabric. A spheroid includes a three-dimensional (3D) cellular structure that arises when cells self-assemble in an environment where they are prevented from adhering to a flat surface. The resulting structure closely resembles the architecture and functionality of tissues in vivo, compared to traditional two-dimensional (2D) cell cultures. Spheroids can be single cell or multicellular. An organoid, by comparison, includes a miniaturized and simplified version of an organ, typically produced in vitro in a 3D culture system from stem cells or progenitor cells. These structures (e.g., organoids) mimic the architecture and functionality of actual organs to a certain extent. Organoids represent a significant advancement over traditional 2D cell cultures and even 3D spheroids because they possess organ- specific cell types and can recapitulate some of the functions of an organ. In some preferred embodiments, cells of the disclosure (including RPE cells) form an organoid. In some embodiments, an organoid formed from a population of cells, including RPE cells, is attached to a substrate suitable for transplantation into a human eye. Substrates suitable for transplantation into an eye include hydrogels, bioengineered collagen matrices, polytetrafluoroethylene (PTFE), human acellular dermal matrix, biocompatible metals and polymers (e.g., titanium, polyimide, and silicone rubber), polyester, silk fibroin, and parylene. Such substrates are discussed above, including hydrogels, polyester, silk fibroin, and parylene. Other substrates may be used. In still other embodiments, assembloids comprising RPE cells of the disclosure are combined with a vascular construct resembling the choricapillaris, the vascular structure at the rear of the eye.

[0098] Methods of Production

[0099] Nucleic Acid Delivery

[0100] Some aspects of the disclosure relate to methods that comprises delivering to a mammalian (e.g., human) retinal pigment epithelial cell an engineered activatable polynucleotide encoding a master regulatory transcription factor (e.g., a polynucleotide comprising an inducible promoter operably linked to an open reading frame encoding a master regulatory transcription factor, such as NGN1, NGN2, NGN3, PAX6, NEURODI, SIX3, or 0TX2). Other aspects of the disclosure relate to methods that comprise delivering to a pluripotent stem cell (e.g., human iPSC or ES cell) an engineered activatable polynucleotide encoding a master regulatory transcription factor (e.g., a polynucleotide comprising an inducible promoter operably linked to an open reading frame encoding a master regulatory transcription factor, such as NGN1, NGN2, NGN3, PAX6, NEURODI, SIX3, or 0TX2). There are various methods of delivering nucleic acids to cells, any of which may be used in accordance with the present disclosure. For example, an engineered activatable polynucleotide may be delivered to a cell using a physical method selected from, e.g., electroporation, microinjection, gene gun (biolistics), sonoporation, and laser-induced transfection; using a viral method selected from, e.g., retroviruses, lentiviruses, adenovirus, adeno-associated viruses, vesicular stomatitis viruses, and herpes simplex viruses; or using other non- viral methods selected from lipid-based transfection, polymer-based transfection, calcium phosphate transfection, dendrimers, nanoparticles, cell-penetrating peptides (CPPs), exosomes, microvesicles, and RNA aptamers.

[0101] In some embodiments, the engineered activatable polynucleotide is delivered to a cell using a physical method. In some embodiments, the physical method is electroporation, microinjection, gene gun (biolistics), sonoporation, or laser-induced transfection. In some embodiments, the physical method is electroporation. In some embodiments, the physical method is microinjection. In some embodiments, the physical method is gene gun (biolistics). In some embodiments, the physical method is sonoporation. In some embodiments, the physical method is laser-induced transfection.

[0102] In some embodiments, the engineered activatable polynucleotide is delivered to a cell using a viral method. In some embodiments, the viral method is retroviruses, lentiviruses, adenovirus, adeno-associated viruses, vesicular stomatitis viruses, or herpes simplex viruses. In some embodiments, the viral method is retroviruses. In some embodiments, the viral method is lentiviruses. In some embodiments, the viral method is adenovirus. In some embodiments, the viral method is adeno-associated viruses. In some embodiments, the viral method is vesicular stomatitis viruses. In some embodiments, the viral method is herpes simplex viruses.

[0103] In some embodiments, the engineered activatable polynucleotide is delivered to a cell using a non-viral method. In some embodiments, the non-viral method is lipid-based transfection, polymer-based transfection, calcium phosphate transfection, dendrimers, nanoparticles, cell-penetrating peptides (CPPs), exosomes, microvesicles, and RNA aptamers. In some embodiments, the non-viral method is lipid-based transfection. In some embodiments, the non-viral method is polymer-based transfection. In some embodiments, the non-viral method is calcium phosphate transfection. In some embodiments, the non-viral method is dendrimers. In some embodiments, the non-viral method is nanoparticles. In some embodiments, the non-viral method is cell-penetrating peptides (CPPs). In some embodiments, the non-viral method is exosomes. In some embodiments, the non-viral method is microvesicles. In some embodiments, the non-viral method is RNA aptamers.

[0104] There are also various methods of targeting an engineered activatable polynucleotide to a specific genomic locus (or loci) in a cell, such as a pluripotent stem cell (e.g., iPSC or ES cell) or an RPE cell at one or more stages of development (e.g., progenitor, immature, or mature). Nonlimiting examples include CRISPR / Cas systems, other programmable nuclease systems such as zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs), restriction enzymes and plasmids, homologous recombination (e.g., using flanking homology arms), and viral vectors (e.g., retroviruses, lentiviruses, and adenoviruses).

[0105] In some embodiments, an engineered activatable polynucleotide is introduced into a safe harbor locus of a pluripotent stem cell (e.g., iPSC or ES cell) or an RPE cell at one or more stages of development (e.g., progenitor, immature, or mature). Safe harbor loci are specific genomic locations where exogenous DNA can be inserted without causing disruptions to endogenous gene function, for example, leading to adverse effects, or causing an oncogenic transformation. These sites are deemed “safe” for gene integration because they minimize the risk of insertional mutagenesis, where the inserted DNA can unintentionally activate or inactivate nearby genes, which could lead to harmful consequences. Non-limiting examples of safe harbor loci include AAVS1, CCR5, ROSA26, Hll, and TRAC.

[0106] Differentiation of PSC to RPE cells

[0107] In some embodiments, a mammalian retinal pigment epithelial cell is derived from a stem cell, such as a pluripotent stem cell (e.g., induced pluripotent stem cell or embryonic stem cell). Methods of differentiating RPE cells from pluripotent stem cells are well known in the art, for example, see Foltz, Leah P, and Dennis O Clegg. “Rapid, Directed Differentiation of Retinal Pigment Epithelial Cells from Human Embryonic or Induced Pluripotent Stem Cells.” Journal of visualized experiments: JoVE ,128 56274. 30 Oct. 2017, doi: 10.3791 / 56274. In some embodiments, methods herein comprise culturing pluripotent stem cells in retinal differentiation media to produce retinal pigment epithelial cells and other neural retinal cell types. Pluripotent stem cells, including iPSCs or ES cells, can be maintained, for example, in base media such as mTeSRl or Essential 8 (E8). These cells can be cultured on matrices such as Matrigel or vitronectin, for example. The conditions for their growth can include a humidified incubator set at 37°C with a 5% CO2 atmosphere. To guide pluripotent stem cells towards a neural fate, they can be transitioned to media that may include DMEM / F12 supplemented with N2 and / or B27, for example. This phase may involve introducing molecules that promote anterior neural differentiation while suppressing other developmental pathways. Commonly added compounds may include but are not limited Noggin and / or SB431542, which inhibit BMP and TGF-beta signaling, respectively. IGF-1 may also be included. DKK1 and / or IWP2 may also be introduced as Wnt inhibitors to further direct the differentiation process. Following neural induction, the culture medium may be modified to further encourage RPE differentiation. This may include, for example, a blend of DMEM / F12, N2, and B27 supplements, in some embodiments, enriched with taurine, hydrocortisone, and / or triiodo-thyronine (T3). Other components may include, for example FGF-basic, Activin A, SU 5402 (FGF receptor- specific tyrosine kinase inhibitor), CHIR99021 (glycogen synthase kinase 3, GSK-3P, inhibitor), and / or dimethyl sulfoxide (DMSO). Over time, one can observe the emergence of pigmented patches within the culture, signaling the onset of RPE differentiation. Once pigmented patches become apparent, they can be carefully dissected and isolated for further culturing, in some embodiments, leading to the maturation and purification of the RPE cells. This may involve transferring these patches to an RPE maintenance medium, for example. This medium can include of DMEM with high glucose, supplemented with components such as serum, N1 supplement, taurine, and / or hydrocortisone. As these cells mature, they typically adopt a characteristic cobblestone appearance upon reaching confluence. In some embodiments, a higher purity of RPE cells can be obtained using additional purification steps. For example, methods such as selective enzymatic treatments or fluorescence- activated cell sorting (FACS) can be employed. The latter may utilize RPE-specific cell-surface markers, such as NCAM1, FOLR1, SLC16A8, or EZR.

[0108] Pluripotent stem cells (e.g., iPSCs or ES cells) may be cultured in media. In some embodiments, the pluripotent stem cells are cultured in differentiation media during a differentiation phase. In some embodiments, the retinal pigment epithelial cells produced in the differentiation phase are further cultured in a maturation phase. For example, see FIG. 1, which is a schematic showing a representative protocol including a differentiation phase and a maturation phase using pluripotent stem cells (e.g., iPSC) cultured in retinal differentiation media (RDM) from Day 0 to Day 14 followed by culturing in maturation media (e.g., X-VIVO™ 15) during a maturation phase that begins on Day 14.

[0109] In some embodiments, pluripotent stem cells (e.g., iPSCs or ES cells) are cultured in differentiation media for multiple days, for example about 1 to about 14 days (e.g., about 10, 11, 12, 13, or 14 days). In some embodiments, the pluripotent stem cells (e.g., iPSCs or ES cells) are cultured in differentiation media for multiple days, for example, no more than 15 days. In some embodiments, the PSCs are cultured in differentiation media for about 1 to about 14 days (e.g., about 1 day). In some embodiments, the PSCs are cultured in differentiation media for about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, or about 14 days. In some embodiments, the PSCs are cultured in differentiation media for about 1 day. In some embodiments, the PSCs are cultured in differentiation media for about 2 days. In some embodiments, the PSCs are cultured in differentiation media for about 3 days. In some embodiments, the PSCs are cultured in differentiation media for about 4 days. In some embodiments, the PSCs are cultured in differentiation media for about 5 days. In some embodiments, the PSCs are cultured in differentiation media for about 6 days. In some embodiments, the PSCs are cultured in differentiation media for about 7 days. In some embodiments, the PSCs are cultured in differentiation media for about 8 days. In some embodiments, the PSCs are cultured in differentiation media for about 9 days. In some embodiments, the PSCs are cultured in differentiation media for about 10 days. In some embodiments, the PSCs are cultured in differentiation media for about 11 days. In some embodiments, the PSCs are cultured in differentiation media for about 12 days. In some embodiments, the PSCs are cultured in differentiation media for about 13 days. In some embodiments, the PSCs are cultured in differentiation media for about 14 days. In some embodiments, the PSCs are cultured in differentiation media for 10 days. In some embodiments, the PSCs are cultured in differentiation media for 11 days. In some embodiments, the PSCs are cultured in differentiation media for 12 days. In some embodiments, the PSCs are cultured in differentiation media for 13 days. In some embodiments, the PSCs are cultured in differentiation media for 14 days.

[0110] In some embodiments, the iPSC-derived retinal pigment epithelial cells have been cultured in differentiation media for no more than about 10-14 days (e.g., 9-14, 9-13, 9-12, 9-11, 9-10, 10-14, 10-13, 10-12, 10-11, 11-14, 11-13, 11-12, 12-14, 12-13, 13-14, days). In some embodiments, the iPSC-derived retinal pigment epithelial cells have been cultured in differentiation media for no more than about 9-14, about 9-13, about 9-12, about 9-11, about 9- 10, about 10-14, about 10-13, about 10-12, about 10-11, about 11-14, about 11-13, about 11-12, about 12-14, about 12-13, about 13-14 days. In some embodiments, the iPSC-derived retinal pigment epithelial cells have been cultured in differentiation media for no more than about 9-15 days. In some embodiments, the iPSC-derived retinal pigment epithelial cells have been cultured in differentiation media for no more than about 9-14 days. In some embodiments, the iPSC- derived retinal pigment epithelial cells have been cultured in differentiation media for no more than about 9-13 days. In some embodiments, the iPSC-derived retinal pigment epithelial cells have been cultured in differentiation media for no more than about 9-12 days. In some embodiments, the iPSC-derived retinal pigment epithelial cells have been cultured in differentiation media for no more than about 9-11 days. In some embodiments, the iPSC-derived retinal pigment epithelial cells have been cultured in differentiation media for no more than about 9-10 days. In some embodiments, the iPSC-derived retinal pigment epithelial cells have been cultured in differentiation media for no more than about 10-14 days. In some embodiments, the iPSC-derived retinal pigment epithelial cells have been cultured in differentiation media for no more than about 10-13 days. In some embodiments, the iPSC-derived retinal pigment epithelial cells have been cultured in differentiation media for no more than about 10-12 days. In some embodiments, the iPSC-derived retinal pigment epithelial cells have been cultured in differentiation media for no more than about 10-11 days. In some embodiments, the iPSC- derived retinal pigment epithelial cells have been cultured in differentiation media for no more than about 11-14 days. In some embodiments, the iPSC-derived retinal pigment epithelial cells have been cultured in differentiation media for no more than about 11-13 days. In some embodiments, the iPSC-derived retinal pigment epithelial cells have been cultured in differentiation media for no more than about 11-12 days. . In some embodiments, the iPSC- derived retinal pigment epithelial cells have been cultured in differentiation media for no more than about 12-14 days. In some embodiments, the iPSC-derived retinal pigment epithelial cells have been cultured in differentiation media for no more than about 12-13 days. In some embodiments, the iPSC-derived retinal pigment epithelial cells have been cultured in differentiation media for no more than about 13-14 days. . In some embodiments, the iPSC- derived retinal pigment epithelial cells have been cultured in differentiation media for no more than 10-14 days.

[0111] In some embodiments, the differentiation media comprises cell culture supplements, such as nicotinamide (NIC). In some embodiments, the differentiation media comprises, alternatively or in addition, growth factors, such as Noggin, Dickkopf WNT Signaling Pathway Inhibitor 1 (DKK1), insulin-like growth factor 1 (IGF-1), fibroblast growth factor (FGF), Activin A, or any combination thereof.

[0112] In some embodiments, the differentiation media is described in Foltz LP et al. J Vis Exp. 2017 Oct 30;(128):56274. In some embodiments, the differentiation media comprises about 5 to about 15 mM NIC (e.g., about 10 mM NIC). In some embodiments, the differentiation media comprises about 5mM, 6mM, 7mM, 8mM, 9mM, lOmM, llmM, 12mM, 13mM, 14mM, 15mM NIC. In some embodiments, the differentiation media comprises about 5 mM NIC. In some embodiments, the differentiation media comprises about 6 mM NIC. In some embodiments, the differentiation media comprises about 7 mM NIC. In some embodiments, the differentiation media comprises about 8 mM NIC. In some embodiments, the differentiation media comprises about 9 mM NIC. In some embodiments, the differentiation media comprises about 10 mM NIC. In some embodiments, the differentiation media comprises about 11 mM NIC. In some embodiments, the differentiation media comprises about 12 mM NIC. In some embodiments, the differentiation media comprises about 13 mM NIC. In some embodiments, the differentiation media comprises about 14 mM NIC. In some embodiments, the differentiation media comprises about 15 mM NIC. In some embodiments, the differentiation media comprises 10 mM NIC.

[0113] In some embodiments, the differentiation media comprises about 25 to about 75 ng / mL noggin or other chemical inhibitor of BMP signaling (e.g., about 50 ng / mL noggin or dorsomorphin). In some embodiments, the differentiation media comprises about 25 ng / mL, 30 ng / mL, 35 ng / mL, 40 ng / mL, 45 ng / mL, 50 ng / mL, 55 ng / mL, 60 ng / mL, 65 ng / mL, 70 ng / mL, 75 ng / mL noggin or dorsomorphin. In some embodiments, the differentiation media comprises about 25 ng / mL noggin or dorsomorphin. In some embodiments, the differentiation media comprises about 30 ng / mL noggin or dorsomorphin. In some embodiments, the differentiation media comprises about 35 ng / mL noggin or dorsomorphin. In some embodiments, the differentiation media comprises about 40 ng / mL noggin or dorsomorphin. In some embodiments, the differentiation media comprises about 45 ng / mL noggin or dorsomorphin. In some embodiments, the differentiation media comprises about 50 ng / mL noggin or dorsomorphin. In some embodiments, the differentiation media comprises about 55 ng / mL noggin or dorsomorphin. In some embodiments, the differentiation media comprises about 60 ng / mL noggin or dorsomorphin. In some embodiments, the differentiation media comprises about 65 ng / mL noggin or dorsomorphin. In some embodiments, the differentiation media comprises about 70 ng / mL noggin or dorsomorphin. In some embodiments, the differentiation media comprises about 75 ng / mL noggin or dorsomorphin. In some embodiments, the differentiation media comprises 50 ng / mL noggin or dorsomorphin.

[0114] In some embodiments, the differentiation media comprises about 5 to about 15 ng / mL DKK-1 or other chemical inhibitor of WNT signaling (e.g., about 10 ng / mL DKK-1 or IWP2). In some embodiments, the differentiation media comprises about 5ng / mL, 6ng / mL, 7ng / mL, 8ng / mL, 9ng / mL, lOng / mL, llng / mL, 12ng / mL, 13ng / mL, 14ng / mL, 15ng / mL DKK-l OR IWP2. In some embodiments, the differentiation media comprises about 5 ng / mL DKK-1 OR

[0115] IWP2. In some embodiments, the differentiation media comprises about 6 ng / mL DKK-1 OR

[0116] IWP2. In some embodiments, the differentiation media comprises about 7 ng / mL DKK-1 OR

[0117] IWP2. In some embodiments, the differentiation media comprises about 8 ng / mL DKK-1 OR

[0118] IWP2. In some embodiments, the differentiation media comprises about 9 ng / mL DKK-1 OR

[0119] IWP2. In some embodiments, the differentiation media comprises about 10 ng / mL DKK-1 OR

[0120] IWP2. In some embodiments, the differentiation media comprises about 11 ng / mL DKK-1 OR

[0121] IWP2. In some embodiments, the differentiation media comprises about 12 ng / mL DKK-1 OR

[0122] IWP2. In some embodiments, the differentiation media comprises about 13 ng / mL DKK-1 OR

[0123] IWP2. In some embodiments, the differentiation media comprises about 14 ng / mL DKK-1 OR

[0124] IWP2. In some embodiments, the differentiation media comprises about 15 ng / mL DKK-1 OR

[0125] IWP2. In some embodiments, the differentiation media comprises 10 ng / mL DKK-1 OR IWP2.

[0126] In some embodiments, the differentiation media comprises about 5 to about 15 ng / mL IGF-1 (e.g., about 10 ng / mL IGF-1). In some embodiments, the differentiation media comprises about 5ng / mL, 6ng / mL, 7ng / mL, 8ng / mL, 9ng / mL, lOng / mL, llng / mL, 12ng / mL, 13ng / mL, 14ng / mL, 15ng / mL IGF-1. In some embodiments, the differentiation media comprises about 5 ng / mL IGF-1. In some embodiments, the differentiation media comprises about 6 ng / mL IGF-1. In some embodiments, the differentiation media comprises about 7 ng / mL IGF-1. In some embodiments, the differentiation media comprises about 8 ng / mL IGF-1. In some embodiments, the differentiation media comprises about 9 ng / mL IGF-1. In some embodiments, the differentiation media comprises about 10 ng / mL IGF-1. In some embodiments, the differentiation media comprises about 11 ng / mL IGF-1. In some embodiments, the differentiation media comprises about 12 ng / mL IGF-1. In some embodiments, the differentiation media comprises about 13 ng / mL IGF-1. In some embodiments, the differentiation media comprises about 14 ng / mL IGF-1. In some embodiments, the differentiation media comprises about 15 ng / mL IGF-1. In some embodiments, the differentiation media comprises 10 ng / mL IGF-1.

[0127] In some embodiments, the differentiation media comprises about 1 to about 10 ng / mL FGF-basic (e.g., about 5 ng / mL FGF-basic). In some embodiments, the differentiation media comprises about 1 ng / mL, about 2 ng / mL, about 3 ng / mL, about 4 ng / mL, about 5 ng / mL, about 6 ng / mL, about 7 ng / mL, about 8 ng / mL, about 9 ng / mL, about 10 ng / mL FGF-basic. In some embodiments, the differentiation media comprises about 1 ng / mL FGF-basic. In some embodiments, the differentiation media comprises about 2 ng / mL FGF-basic. In some embodiments, the differentiation media comprises about 3 ng / mL FGF-basic. In some embodiments, the differentiation media comprises about 4 ng / mL FGF-basic. In some embodiments, the differentiation media comprises about 5 ng / mL FGF-basic. In some embodiments, the differentiation media comprises about 6 ng / mL FGF-basic. In some embodiments, the differentiation media comprises about 7 ng / mL FGF-basic. In some embodiments, the differentiation media comprises about 8 ng / mL FGF-basic. In some embodiments, the differentiation media comprises about 9 ng / mL FGF-basic. In some embodiments, the differentiation media comprises about 10 ng / mL FGF-basic. In some embodiments, the differentiation media comprises 5 ng / mL FGF-basic.

[0128] In some embodiments, the differentiation media comprises about 25 to about 500 ng / mL activin A (e.g., about 100 ng / mL activin A). In some embodiments, the differentiation media comprises about 25 ng / mL, about 50 ng / mL, about 100 ng / mL, about 250 ng / mL, about 500 ng / mL activin A. In some embodiments, the differentiation media comprises about 25 ng / mL activin A. In some embodiments, the differentiation media comprises about 50 ng / mL activin A. In some embodiments, the differentiation media comprises about 100 ng / mL activin A. In some embodiments, the differentiation media comprises about 250 ng / mL activin A. In some embodiments, the differentiation media comprises about 500 ng / mL activin A. In some embodiments, the differentiation media comprises 100 ng / mL activin A.

[0129] In some embodiments, the differentiation media comprises about 5-10 mM NIC, about 25-75 ng / mL noggin, about 5-15 ng / mL DKK-1, about 5-15 ng / mL IGF-1, or any combination thereof. In some embodiments, the differentiation media comprises about 10 mM NIC, about 50 ng / mL noggin, about 10 ng / mL DKK-1, about 10 ng / mL IGF-1, or any combination thereof.

[0130] In some embodiments, the differentiation media comprises, alternatively or in addition, protein inhibitors, such the tyrosine kinase inhibitor SU 5402, the glycogen synthase kinase 3 inhibitor CHIR99021, or both.

[0131] In some embodiments, the differentiation media comprises FGF receptor- specific tyrosine kinase inhibitor, SU 5402. In some embodiments, the differentiation media comprises SU 5402 in a concentration of about 5 to about 15 pM (e.g., about 5, 7, 8, 9, 10, 11, 12, 13, 14, 15 pM). In some embodiments, the differentiation media comprises SU 5402 in a concentration of about 5 pM, 6 pM, 7 pM, 8 pM, 9 pM, 10 pM, 11 pM, 12 pM, 13 pM, 14 pM, 15 pM. In some embodiments, the differentiation media comprises SU 5402 in a concentration of about 5 pM. In some embodiments, the differentiation media comprises SU 5402 in a concentration of about 6 pM. In some embodiments, the differentiation media comprises SU 5402 in a concentration of about 7 M. In some embodiments, the differentiation media comprises SU 5402 in a concentration of about 8 pM. In some embodiments, the differentiation media comprises SU 5402 in a concentration of about 9 p M. In some embodiments, the differentiation media comprises SU 5402 in a concentration of about 10 pM. In some embodiments, the differentiation media comprises SU 5402 in a concentration of about 11 pM. In some embodiments, the differentiation media comprises SU 5402 in a concentration of about 12 pM. In some embodiments, the differentiation media comprises SU 5402 in a concentration of about 13 pM. In some embodiments, the differentiation media comprises SU 5402 in a concentration of about 14 pM. In some embodiments, the differentiation media comprises SU 5402 in a concentration of about 15 pM. In some embodiments, the differentiation media comprises SU 5402 in a concentration of 10 pM.

[0132] In some embodiments, the differentiation media comprises CHIR99021 in a concentration of about 0.5 to about 10 pM (e.g., about 3 pM). In some embodiments, the differentiation media comprises CHIR99021 in a concentration of about 0.5 pM, about 1 pM, about 1.5 pM, about 3 pM, about 5 pM, about 10 pM. In some embodiments, the differentiation media comprises CHIR99021 in a concentration of about 0.5 pM. In some embodiments, the differentiation media comprises CHIR99021 in a concentration of about 1 pM. In some embodiments, the differentiation media comprises CHIR99021 in a concentration of about 1.5 pM. In some embodiments, the differentiation media comprises CHIR99021 in a concentration of about 3 pM. In some embodiments, the differentiation media comprises CHIR99021 in a concentration of about 5 pM. In some embodiments, the differentiation media comprises CHIR99021 in a concentration of about 10 pM. In some embodiments, the differentiation media comprises CHIR99021 in a concentration of 3 pM.

[0133] In some embodiments, the differentiation media comprises 5-15 mM NIC, 25-75 ng / mL noggin, 5-15 ng / mL DKK-1, and 5-15 ng / mL IGF-1. In some embodiments, the differentiation media comprises 5-15 mM NIC, 1-10 ng / mL FGF-basic, 5-15 ng / mL noggin, 5-15 ng / mL DKK- 1, and 5-15 ng / mL IGF-1. In some embodiments, the differentiation media comprises 50-150 ng / mL activin A, 5-10 ng / mL DKK-1, and 5-10 ng / mL IGF-1. In some embodiments, the differentiation media comprises 50-150 ng / mL activin A and 5-15 pM SU 5402. In some embodiments, the differentiation media comprises 50-150 ng / mL activin A, 5-15 pM SU 5402, and 1-10 pM CHIR99021. In some embodiments, the differentiation media comprises 10 mM NIC, 50 ng / mL noggin, 10 ng / mL DKK-1, and 10 ng / mL IGF-1. In some embodiments, the differentiation media comprises 10 mM NIC, 5 ng / mL FGF-basic, 10 ng / mL noggin, 10 ng / mL DKK-1, and 10 ng / mL IGF-1. In some embodiments, the differentiation media comprises 100 ng / mL activin A, 10 ng / mL DKK-1, and 10 ng / mL IGF-1. In some embodiments, the differentiation media comprises 100 ng / mL activin A and 10 pM SU 5402. In some embodiments, the differentiation media comprises 100 ng / mL activin A, 10 p M SU 5402, and 3 pM CHIR99021.

[0134] In some embodiments, on Day 0 of the culturing, the differentiation media comprises NIC, Noggin, DKK1, IGF-1, or a combination thereof. In some embodiments, on Day 0 of the culturing, the differentiation media comprises 5-10 mM NIC, 25-75 ng / mL noggin, 5-10 ng / mL DKK-1, and 5-10 ng / mL IGF-1. In some embodiments, on Day 1 of the culturing, the differentiation media comprises NIC, Noggin, DKK1, IGF-1, or a combination thereof. In some embodiments, on Day 1 of the culturing, the differentiation media comprises 5-10 mM NIC, 25- 75 ng / mL noggin, 5-10 ng / mL DKK-1, and 5-10 ng / mL IGF-1. In some embodiments, on Day 2 and / or 3 of the culturing, the differentiation media comprises NIC, Noggin, DKK1, IGF-1, FGF, or a combination thereof. In some embodiments, on Day 2 and / or 3 of the culturing, the differentiation media comprises 5-10 mM NIC, 1-10 ng / mL FGF-basic, 5-10 ng / mL noggin, 5-10 ng / mL DKK-1, and 5-10 ng / mL IGF-1. In some embodiments, on Day 4 and / or 5 of the culturing, the differentiation media comprises Activin A, DKK1, IGF-1, or a combination thereof. In some embodiments, on Day 4 and / or 5 of the culturing, the differentiation media comprises 50-150 ng / mL activin A, 5-10 ng / mL DKK-1, and 5-10 ng / mL IGF-1. In some embodiments, on Day 6 and / or 7 of the culturing, the differentiation media comprises Activin A, SU 5402, or both. In some embodiments, on Day 6 and / or 7 of the culturing, the differentiation media comprises 50-150 ng / mL activin A and 5-10 pM SU 5402. In some embodiments, on Day 8, 9, 10, 11, and / or 12 of the culturing, the differentiation media comprises Activin A, SU 5402, CHIR99021, or a combination thereof (see, e.g., FIG. 1). In some embodiments, on Day 8, 9, 10, 11, and / or 12 of the culturing, the differentiation media comprises 50-15-ng / mL activin A, 5- 10 pM SU 5402, and 1-10 pM CHIR99021.

[0135] In some embodiments, on Day 0 of the culturing, the differentiation media comprises NIC, Noggin, DKK1, IGF-1, or a combination thereof. In some embodiments, on Day 0 of the culturing, the differentiation media comprises 10 mM NIC, 50 ng / mL noggin, 10 ng / mL DKK-1, and 10 ng / mL IGF-1. In some embodiments, on Day 1 of the culturing, the differentiation media comprises NIC, Noggin, DKK1, IGF-1, or a combination thereof. In some embodiments, on Day 1 of the culturing, the differentiation media comprises 10 mM NIC, 50 ng / mL noggin, 10 ng / mL DKK-1, and 10 ng / mL IGF-1. In some embodiments, on Day 2 and / or 3 of the culturing, the differentiation media comprises NIC, Noggin, DKK1, IGF-1, FGF, or a combination thereof. In some embodiments, on Day 2 and / or 3 of the culturing, the differentiation media comprises 10 mM NIC, 5 ng / mL FGF-basic, 10 ng / mL noggin, 10 ng / mL DKK-1, and 10 ng / mL IGF-1. In some embodiments, on Day 4 and / or 5 of the culturing, the differentiation media comprises Activin A, DKK1, IGF-1, or a combination thereof. In some embodiments, on Day 4 and / or 5 of the culturing, the differentiation media comprises 100 ng / mL activin A, 10 ng / mL DKK-1, and 10 ng / mL IGF-1. In some embodiments, on Day 6 and / or 7 of the culturing, the differentiation media comprises Activin A, SU 5402, or both. In some embodiments, on Day 6 and / or 7 of the culturing, the differentiation media comprises 100 ng / mL activin A and 10 pM SU 5402. In some embodiments, on Day 8, 9, 10, 11, and / or 12 of the culturing, the differentiation media comprises Activin A, SU 5402, CHIR99021, or a combination thereof (see, e.g., FIG. 1). In some embodiments, on Day 8, 9, 10, 11, and / or 12 of the culturing, the differentiation media comprises 100 ng / mL activin A, 10 p M SU 5402, and 3 pM CHIR99021.

[0136] Maturation of RPE cells

[0137] In some embodiments, the retinal pigment epithelial cells produced in the differentiation phase are further cultured in a maturation phase. For example, see FIG. 1, which is a schematic showing a representative protocol including a differentiation phase and a maturation phase using pluripotent stem cells (e.g., iPSC) cultured in retinal differentiation media (RDM) to produce mature RPE cells that are further culture in a maturation phase that begins on Day 14.

[0138] In some embodiments, the retinal pigment epithelial cells (e.g., mature-derived RPE cells) (e.g., mature-derived RPE cells) are cultured in maturation media (e.g., X-VIVO™ 15) for about 1-100 (e.g., 1-100, 1-90, 1-60, 1-30, 28-63 30-100, 30-90, 30-60, 60-100, 60-90, or 90- 100) days. In some embodiments, the retinal pigment epithelial cells (e.g., mature-derived RPE cells) are cultured in maturation media (e.g., X-VIVO™ 15) for about 1-100, 1-90, 1-60, 1-30, 28-63, 30-100, 30-90, 30-60, 60-100, 60-90, or 90-100 days. In some embodiments, the retinal pigment epithelial cells (e.g., mature-derived RPE cells) are cultured in maturation media (e.g., X-VIVO™ 15) for about 1 day. In some embodiments, the retinal pigment epithelial cells (e.g., mature-derived RPE cells) are cultured in maturation media (e.g., X-VIVO™ 15) for about 28 days. In some embodiments, the retinal pigment epithelial cells (e.g., mature-derived RPE cells) are cultured in maturation media (e.g., X-VIVO™ 15) for about 30 days. In some embodiments, the retinal pigment epithelial cells (e.g., mature-derived RPE cells) are cultured in maturation media (e.g., X-VIVO™ 15) for about 60 days. In some embodiments, the retinal pigment epithelial cells (e.g., mature-derived RPE cells) are cultured in maturation media (e.g., X- VIVO™ 15) for about 63 days. In some embodiments, the retinal pigment epithelial cells (e.g., mature-derived RPE cells) are cultured in maturation media (e.g., X-VIVO™ 15) for about 90 days. In some embodiments, the retinal pigment epithelial cells (e.g., mature-derived RPE cells) are cultured in maturation media (e.g., X-VIVO™ 15) for about 100 days. In some embodiments, the retinal pigment epithelial cells (e.g., mature-derived RPE cells) are cultured in maturation media (e.g., X-VIVO™ 15) for 28-63 days. In some embodiments, the retinal pigment epithelial cells (e.g., mature-derived RPE cells) are cultured in maturation media (e.g., X-VIVO™ 15) for at least 100 days.

[0139] In some embodiments the retinal pigment epithelial cells (e.g., mature-derived RPE cells) are cultured in maturation media. In some embodiments the maturation media is X-VIVO™ 15 media. In some embodiments, the X-VIVO™ 15 media is supplemented with 10 pM Y-27632. In some embodiments, the maturation media comprises Y-27632 in a concentration of about 10 (e.g., 5, 7, 8, 9, 10, 11, 12, 13, 14, 15) pM. In some embodiments, the maturation media (e.g., X- VIVO™ 15) comprises Y-27632 in a concentration of about 5 pM, 6 pM, 7 pM, 8 pM, 9 pM, 10 pM, 11 pM, 12 pM, 13 pM, 14 pM, 15 pM. In some embodiments, the maturation media (e.g., X-VIVO™ 15) comprises Y-27632 in a concentration of about 5 pM. In some embodiments, the maturation media (e.g., X-VIVO™ 15) comprises Y-27632 in a concentration of about 6 pM. In some embodiments, the maturation media (e.g., X-VIVO™ 15) comprises Y-27632 in a concentration of about 7 pM. In some embodiments, the maturation media (e.g., X-VIVO™ 15) comprises Y-27632 in a concentration of about 8 pM. In some embodiments, the maturation media (e.g., X-VIVO™ 15) comprises Y-27632 in a concentration of about 9 pM. In some embodiments, the maturation media (e.g., X-VIVO™ 15) comprises Y-27632 in a concentration of about 10 pM. In some embodiments, the maturation media (e.g., X-VIVO™ 15) comprises Y- 27632 in a concentration of about 11 pM. In some embodiments, the maturation media (e.g., X- VIVO™ 15) comprises Y-27632 in a concentration of about 12 pM. In some embodiments, the maturation media (e.g., X-VIVO™ 15) comprises Y-27632 in a concentration of about 13 pM. In some embodiments, the maturation media (e.g., X-VIVO™ 15) comprises Y-27632 in a concentration of about 14 pM. In some embodiments, the maturation media (e.g., X-VIVO™ 15) comprises Y-27632 in a concentration of about 15 pM. In some embodiments, the maturation media (e.g., X-VIVO™ 15) comprises Y-27632 in a concentration of 10 pM. Culture Systems

[0140] Methods herein, in some aspects, comprise culturing retinal pigment epithelial cells in cell culture media, for example, on transwell filters in two dimensions, or in a -three-dimensional culture. Three-dimensional (3D) cell culture systems generally aim to more closely mimic an in vivo cellular environment, compared to traditional two-dimensional (2D) cultures on flat surfaces. In 3D cultures, cells grow in all directions, creating cell-cell and cell-matrix interactions that are more representative of natural tissue structures. To achieve this, a variety of scaffolds and matrices have been developed, for example: natural scaffolds, such as collagen, Matrigel, alginate, agarose, silk fibroin, chitosan, fibrin, and hyaluronic acid; and synthetic scaffolds, such as poly(lactic-co-glycolic acid) (PLGA), polyethylene glycol (PEG), polycaprolactone (PCL), and polyacrylamide. Composite and hybrid scaffolds have also been developed, which combine natural and synthetic materials, for example, collagen with synthetic polymers. Scaffold-free systems may also be used in 3D cultures to develop, for example, spheroids (e.g., using hanging drop plates or low-attachment surfaces), organoids, and cell sheets (e.g., cells are cultured in 2D to form confluent sheets, which can then be stacked to create 3D structures). It should be understood, however, that spheroids and organoids can also be produced in a 3D culture system that uses a scaffold.

[0141] The product of culturing RPE cells in a sheet or a 3D culture system, such as a spheroid or organoid, may be attached to a substrate suitable for transplantation into a human eye, including, for example, hydrogels, polyester, silk fibroin, and parylene. This attachment occurs, in some embodiments, by simply culturing the RPE cells in the presence of the substrate. Thus, in some embodiments, the scaffold using in a 3D culture is or includes a substrate suitable for transplantation into a human eye.

[0142] In some embodiments, the present disclosure relates to a bilayer culture. As described herein, the term “bilayer culture” includes a cell culture system where cells are grown in two layers of cells. In some embodiments, the bilayer culture comprises a first population of retinal pigment epithelial (RPE) cells and a second population of RPE cells. In some embodiments, the first population of RPE cells and the second population of RPE cells are in separate layers of the bilayer culture. In some embodiments, the first population of the RPE cells comprises wild-type RPE cells. In some embodiments, the second population of the RPE cells comprises wild-type RPE cells. In some embodiments, the first population of the RPE cells comprises RPE cells comprising a polynucleotide having an inducible promoter operably linked to an open reading frame encoding a master regulatory transcription factor. In some embodiments, the second population of the RPE cells comprises RPE cells comprising a polynucleotide having an inducible promoter operably linked to an open reading frame encoding a master regulatory transcription factor. In some embodiments, the first population of the RPE cells comprises RPE cells and the second population of RPE cells comprises a polynucleotide having an inducible promoter operably linked to an open reading frame encoding a master regulatory transcription factor. In some embodiments, the second population of the RPE cells comprises RPE cells and the first population of RPE cells comprises a polynucleotide having an inducible promoter operably linked to an open reading frame encoding a master regulatory transcription factor.

[0143] In some embodiments, the first population of RPE cells and the second population of RPE cells are in a ratio of about 35:65, about 40:60, about 45:55, or about 50:50. In some embodiments, the first population of RPE cells and the second population of RPE cells are in a ratio of about 35:65, 40:60, 45:55, 50:50. In some embodiments, the first population of RPE cells and the second population of RPE cells are in a ratio of 50:50.

[0144] Some aspects of the present disclosure relate to a method of treating a retinal disease comprising, administering to a subject having a retinal disease the bilayer culture of the present disclosure and a substrate suitable for transplantation into an eye.

[0145] The cultivation of retinal organoids may start with, for example, culturing RPE cells in a base medium, for example, DMEM / F12 (Dulbecco's Modified Eagle Medium / Nutrient Mixture F-12) or Neurobasal medium. In some embodiments, these media form the foundation for the addition of further supplements and growth factors that will guide the differentiation process. As differentiation proceeds, serum replacements may be added to the base medium. B-27 and N-2, for example, are two such supplements that provide essential nutrients to the developing organoids. They can effectively replace serum, in some embodiments, ensuring a more defined culture condition. Additionally, antibiotics such as penicillin- streptomycin may be incorporated to prevent bacterial contamination. Other additives, such as GlutaMAX (a stable form of L- glutamine) an / or serum, can play a role in the early stages of differentiation. The ROCK inhibitor, Y- 27632, may also, in some embodiments, be included to promote cell survival, especially during cell passaging. Driving cells towards retinal differentiation typically necessitates a series of growth factors and molecules. These may be added in a sequence or at specific stages. Noggin, an antagonist of BMP signaling, DKK1, IWP2 (Wnt pathway inhibitors), and SB431542 (an inhibitor of TGF-beta signaling) are common additions, for example. Cell culture media (e.g., differentiation media or maturation media) in which RPE cells are cultured may comprise, for example, one or more growth factors selected from NIC, Noggin, DKK1, IGF-1, Activin A, SU 5402, and CHIR99021. NIC (nicotinamide) is a derivative of vitamin B3 and is the primary precursor of nicotinamide adenine dinucleotide (NAD+), a coenzyme that is central to cellular metabolism. Noggin is a protein involved in the development of multiple tissues and acts as an inhibitor of multiple bone morphogenetic proteins (BMPs). DKK1 (Dickkopf WNT Signaling Pathway Inhibitor 1) is a secreted inhibitor of the canonical (P-catenin) Wnt pathway. IGF-1 (insulin-like growth factor 1) is a hormone and is the primary mediator by which growth hormone (GH) influences development. Activin A is a member of the transforming growth factor b (TGF-b) superfamily and a pluripotent factor important for cell differentiation, proliferation, remodeling, and morphogenesis. SU 5402 is a tyrosine kinase inhibitor that targets vascular endothelial growth factor receptor (VEGFR), fibroblast growth factor receptor (FGFR), and platelet-derived growth factor receptor-beta (PDGF-RP). CHIR99021 is an aminopyrimidine derivative that inhibits glycogen synthase kinase (GSK) 3 and can bias stem cell differentiation to particular cell lineages.

[0146] In some embodiments, cell culture media (e.g., differentiation media) comprises NIC. In some embodiments, cell culture media (e.g., differentiation media) comprises Noggin. In some embodiments, cell culture media (e.g., differentiation media) comprises DKK1. In some embodiments, cell culture media (e.g., differentiation media) comprises IGF-1. In some embodiments, cell culture media (e.g., differentiation media) comprises Activin A. In some embodiments, cell culture media (e.g., differentiation media) comprises SU 5402. In some embodiments, cell culture media (e.g., differentiation media) comprises CHIR99021. In some embodiments, cell culture media (e.g., differentiation media) comprises transforming growth factor beta (TGF-P). In some embodiments, cell culture media (e.g., differentiation media) comprises Wnt signaling inhibitor casein kinase inhibitor (CKI)-7. In some embodiments, cell culture media (e.g., differentiation media) comprises the TGF-P ligand Eefty-A. In some embodiments, cell culture media (e.g., differentiation media and / or maturation media) comprises the fibroblast growth factor antagonist Y-27632. In some embodiments, cell culture media (e.g., differentiation media) comprises the nodal signaling inhibitor SB431542. Other factors, inhibitors, and cell culture supplements may be included in the cell culture media in which RPE cells are cultured. Exemplary methods for culturing RPE cells include, for example, Michelet et al., Stem Cell Res Ther, 2020. 11(1):47; Foltz and Clegg, J Vis Exp, 2017. 128:56274; and Vaajasaari et al., Mol Vis, 2011. 17:558-575. Retinal pigment epithelium cells of the disclosure may be cultured in cell culture media for about a week (about 7 days) to about a month (about 30 days), for example. In some embodiments, RPE cells are cultured for about 10 to 25 days. In some embodiments, RPE cells are cultured for about 15 to 25 days. In some embodiments, RPE cells are cultured for about 7, about 10, about 15, about 20, about 25, or about 30 days. In some embodiments, RPE cells are cultured for about 20 days. In some embodiments, retinal pigment epithelium cells of the disclosure may be cultured in cell culture media for about a week (about 7 days) to about 90 days, for example, for mature cell types. In some embodiments, RPE cells are cultured for about 30 to 90 days, about 30 to 60 days, or about 60 to 90 days. In some embodiments, RPE cells are cultured for about 30, about 40, about 50, about 60, about 70, about 80, or about 90 days.

[0147] Following delivery of an engineered activatable polynucleotide to a pluripotent stem cell (e.g., iPSC or ES cell) or an RPE cell, expression of the master regulatory transcription factor encoded by the polynucleotide may be activated. Activation will largely depend on the type of activatable system used. For example, if a polynucleotide includes an inducible promoter, then the cognate inducing agent should be used to activate expression of an open reading frame. For example, with a doxycycline (Dox) system, activation may include culturing transfected RPE cells in the presence of Dox (e.g., adding Dox to the culture media), or otherwise contacting RPE cells with Dox (e.g., administering Dox to a subject who has received an RPE implant). Likewise, for heat- sensitive systems, exposing the RPE cells to heat will activate (or repress) expression, and for light-sensitive systems, exposing the RPE cells to light will activate (or repress) expression. A skilled artisan can determine the inducing agent for any particular inducible (or other activatable) gene expression system.

[0148] In some embodiments, doxycycline is added to the culture media (e.g., differentiation media and / or maturation media). In some embodiments, about 100-2000 (e.g., 100-2000, 100- 1500, 100-1000, 100-500, 100-250, 250-2000, 250-1500, 250-1000, 250-500, 500-2000, 500- 1500, 500-1000, 1000-2000, 1000-1500, 1500-2000) ng of doxycycline is added to the culture media (e.g., differentiation media and / or maturation media). In some embodiments, about 100- 2000, 100-1500, 100-1000, 100-500, 100-250, 250-2000, 250-1500, 250-1000, 250-500, 500- 2000, 500-1500, 500-1000, 1000-2000, 1000-1500, 1500-2000 ng of doxycycline is added to the culture media (e.g., differentiation media and / or maturation media). In some embodiments, about 100, 250, 500, 1000, 1500, 2000 ng of doxycycline is added to the culture media (e.g., differentiation media and / or maturation media). In some embodiments, about 100 ng of doxycycline is added to the culture media (e.g., differentiation media and / or maturation media). In some embodiments, about 250 ng of doxycycline is added to the culture media (e.g., differentiation media and / or maturation media). In some embodiments, about 500 ng of doxycycline is added to the culture media (e.g., differentiation media and / or maturation media).In some embodiments, about 1000 ng of doxycycline is added to the culture media (e.g., differentiation media and / or maturation media). In some embodiments, about 1500 ng of doxycycline is added to the culture media (e.g., differentiation media and / or maturation media). In some embodiments, about 2000 ng of doxycycline is added to the culture media (e.g., differentiation media and / or maturation media). In some embodiments, 200 mg of doxycycline is added to the culture media (e.g., differentiation media and / or maturation media).

[0149] In some embodiments, doxycycline is administered every 12 hours for about 24-120 (e.g., 24-120, 24-96, 24-72, 24-48, 48-120, 48-96, 48-72, 72-120, 72-96, 96-120) hours. In some embodiments, doxycycline is administered every 12 hours for about 24-120, 24-96, 24-72, 24- 48, 48-120, 48-96, 48-72, 72-120, 72-96, 96-120 hours. In some embodiments, doxycycline is administered every 12 hours for about 24-120 hours. In some embodiments, doxycycline is administered every 12 hours for about 24, 48, 72, 96, 120 hours. In some embodiments, doxycycline is administered every 12 hours for about 24 hours. In some embodiments, doxycycline is administered every 12 hours for about 48 hours. In some embodiments, doxycycline is administered every 12 hours for about 72 hours. In some embodiments, doxycycline is administered every 12 hours for about 96 hours. In some embodiments, doxycycline is administered every 12 hours for about 120 hours.

[0150] In some embodiments, doxycycline is administered every 12 hours for about 24-120 hours at a dose of about 200 (e.g., 100, 150, 200, 250) mg. In some embodiments, doxycycline is administered every 12 hours for about 24-120 hours at a dose of about 100, 150, 200, 250 mg. In some embodiments, doxycycline is administered every 12 hours for about 24-120 hours at a dose of about 100 mg. In some embodiments, doxycycline is administered every 12 hours for about 24-120 hours at a dose of about 150 mg. In some embodiments, doxycycline is administered every 12 hours for about 24-120 hours at a dose of about 200 mg. In some embodiments, doxycycline is administered every 12 hours for about 24-120 hours at a dose of about 250 mg. In some embodiments, doxycycline is administered every 12 hours for about 24-120 hours at a dose of 200 mg. Methods of Treatment

[0151] Various aspects of the disclosure provide methods of treating a subject having a degenerative eye disease. Degenerative eye diseases are a group of disorders that result in the progressive loss of vision due to the gradual deterioration of eye structures. Non-limiting examples of degenerative eye diseases include age-related macular degeneration (dry (atrophic) or wet (neovascular)), glaucoma, retinopathy, retinitis pigmentosa, Stargardt disease, optic neuropathies, and corneal dystrophies. In some embodiments, a subject has (e.g., is diagnosed with) age-related macular degeneration.

[0152] As described herein, a “subject” or “subject in need thereof’ includes a subject in need of treatment for an eye disorder. In some embodiments, the eye disorder is a retinal disease or a degenerative eye disease. In some embodiments, the eye disorder is characterized by loss of photoreceptors. Non-limiting examples of eye disorders (e.g., retinal disease or a degenerative eye disease) include age-related macular degeneration, glaucoma, retinopathy, retinitis pigmentosa, Stargardt disease, optic neuropathies, and corneal dystrophies. In some embodiments, the eye disorder (e.g., retinal disease or a degenerative eye disease) is age-related macular degeneration. In some embodiments, the subject in need may be one that is “non- responsive” or “refractory” to a standard therapy for the degenerative eye disease (e.g., age- related macular degeneration, glaucoma, retinopathy, retinitis pigmentosa, Stargardt disease, optic neuropathies, and corneal dystrophies). In some embodiments, the terms “non-responsive” and “refractory” refers to the subject's response to therapy as not clinically adequate to relieve one or more symptoms associated with the degenerative eye disease.

[0153] A subject generally refers to a mammal. The mammal may be, for example, a human, primate, mouse, rat, dog, cat, cow, horse, goat, camel, sheep, or a pig. In some embodiments, the subject is a primate. In some embodiments, the subject is a primate. In some embodiments, the subject is a human.

[0154] In some embodiments, a subject has degenerative eye disease (e.g., age-related macular degeneration, glaucoma, retinopathy, retinitis pigmentosa, Stargardt disease, optic neuropathies, and corneal dystrophies). In some embodiments, the subject exhibits one or more symptoms of degenerative eye disease (e.g., age-related macular degeneration, glaucoma, retinopathy, retinitis pigmentosa, Stargardt disease, optic neuropathies, and corneal dystrophies).

[0155] In some embodiments, the subject is about 48 to about 95 years of age. In some embodiments, the subject is about 48, about 49, about 50, about 51, about 52, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95 years of age. In some embodiments, the subject is about 48 years of age. In some embodiments, the subject is about 49 years of age. In some embodiments, the subject is about 50 years of age. In some embodiments, the subject is about 51 years of age. In some embodiments, the subject is about 52 years of age. In some embodiments, the subject is about 55 years of age. In some embodiments, the subject is about 60 years of age. In some embodiments, the subject is about 65 years of age. In some embodiments, the subject is about 70 years of age. In some embodiments, the subject is about 75 years of age. In some embodiments, the subject is about 80 years of age. In some embodiments, the subject is about 85 years of age. In some embodiments, the subject is about 90 years of age. In some embodiments, the subject is about 70 years of age. In some embodiments, the subject is about 95 years of age.

[0156] In some embodiments, the subject has an early onset genetic form of macular dystrophy or retinopathy.

[0157] As described herein, “an effective amount” includes an amount that alleviates one or more symptom(s) associated with a particular condition, such as degenerative eye disease (e.g., age-related macular degeneration, glaucoma, retinopathy, retinitis pigmentosa, Stargardt disease, optic neuropathies, and corneal dystrophies).

[0158] Treatment of a particular disease includes the amelioration of one or more symptoms associated with the disease. For example, with respect to age-related macular degeneration, the symptoms in humans include blurred or distorted vision, a dark spot in the center of vision, difficulty recognizing faces, diminished or changed color perception, difficulty adapting to low light levels, straight lines appearing wavy or distorted, decreased intensity or brightness of colors, increased need for brighter light for reading and other activities, and trouble or discomfort with glare. Thus, treatment of AMD using a method of the present disclosure may result in a reduction or lessening of one or more of the foregoing symptoms (e.g., clearer vision, removal of dark spot, improvement in facial recognition, restoration of color perception, etc.). This relative change in symptoms may be compared to “baseline” symptoms, for example, which are symptoms exhibited by as subject prior to the symptomatic onset of the disease.

[0159] Methods of the disclosure comprise administering to an eye (left or right) of a subject a mammalian retinal pigment epithelial cell comprising an engineered activatable polynucleotide encoding a master regulatory transcription factor. Other methods of the disclosure comprise administering to an eye (left or right) of a subject a population or subpopulation of cells (e.g., a retinal graft) comprising a mammalian retinal pigment epithelial cell that comprise an engineered activatable polynucleotide encoding a master regulatory transcription factor. Yet other methods of the disclosure comprise administering to an eye (left or right) of a subject a sheet, spheroid, or organoid including a population or subpopulation of cells comprising a mammalian retinal pigment epithelial cell that comprise an engineered activatable polynucleotide encoding a master regulatory transcription factor. Administration to an eye may include, for example, the use of fine-needle syringes, vitrectomy machines, microscope-guided delivery systems, optical coherence tomography guidance, cannulas, cell carriers (e.g., a biocompatible material, such as a hydrogel, or scaffold implanted into the eye), or gene therapy vectors. The vehicle for delivery will depend in part on the material being delivered - for example, cells or polynucleotides (e.g., expression vectors). In some embodiments, mammalian RPE cells of the disclosure are administered to an eye of a subject using a substrate suitable for transplantation into a human eye, as described elsewhere herein.

[0160] In some aspects, an engineered activatable polynucleotide encoding a master regulatory transcription factor is delivered directly to the eye of a subject (in vivo), for example, to retinal pigment epithelial cells of the subject.

[0161] Following delivery of an engineered activatable polynucleotide to a subject (via transfected RPE cells, RPE cells comprising the engineered activatable polynucleotide, RPE cells differentiated from pluripotent stem cells comprising the engineered activatable polypeptide, or via direct administration to the eye) the polynucleotide may be activated, as described elsewhere herein. For example, if the engineered activatable polynucleotide includes an inducible promoter operably linked to an open reading frame, then a method may include administering an inducing agent (e.g., dox) to a subject (e.g., to the eye of the subject in which the engineered activatable polynucleotide was delivered).

[0162] EXAMPLES

[0163] Example 1. Induction of Neurogenin-2 (NGN2) in human retinal pigment epithelial (RPE) cells upregulates photoreceptor and early eye lineage markers.

[0164] A human induced pluripotent stem cell (hiPSC) line that is engineered to express Neurogenin-2 (NGN2), a master regulatory transcription factor in central nervous system development, under control of the Tetracycline inducible expression (TET) promoter (NGN K0LF2.1 cells). In studies preliminary to that described in this Example, it was confirmed that activation of NGN2 in hiPSCs resulted in NGN2 expression and induction of cortical neurons. Here, hiPSCs were differentiated into RPE cells and cultured for 20 days, at which point doxycycline was added to induce NGN2 expression (FIG. 1). Cells remained in culture for 16 more days after NGN2 induction, before being fixed and stained for markers of RPE cells and photoreceptor lineages (including early eye lineages). Relative to control cells, the proportion of cells in NGN2-induced cultures expressing the RPE markers Microphtalmia-associated transcription factor (MITF), Bestrophin-1 (BEST1), Retinoid isomerohydrolase (RPE65), and Serpin family F member 1 (SERPNF1) was markedly decreased, while the proportion of cells expressing the photoreceptor markers Rhodopsin (RHO), Recoverin (RVCRN), Microtubule associate protein 2 (MAP2), and the homeobox transcription factors CRX and CHX10 was substantially increased. The early eye lineage markers Paired box 6 (PAX6) and Orthodenticle homeobox 2 (0TX2) were also expressed in a higher proportion of NGN2-induced cells (FIGs. 2A-2C). These data indicate that RPE from human pluripotent stem cells can be selectively reprogrammed to give rise to cells of other retinal lineages, such as photoreceptors, as well as immature progenitor cells.

[0165] Methods

[0166] DMEM / F12 Supplement Preparation:

[0167] Make 100 mF of retinal differentiation medium (RDM) by adding 1 mF of lOOx N2 supplement, 2 mF of 50x B27 supplement, and 1 mF of lOOx non-essential amino acid (NEAA) to 96 mF of Dulbecco's modified essential medium / nutrient mixture F12 9 (DMEM / F12) Nicotinamide (NIC) Preparation:

[0168] Make 10 mF of 1 M nicotinamide (NIC) by dissolving 1.221 g of NIC in 8 mF of sterile water, vortexing, and bringing the volume to 10 mF with sterile water. Sterile filter the solution and sore at 4 °C.

[0169] Growth Factors and Small Molecules Preparation: a. Prepare 0.1% bovine serum albumin (BSA) solution by dissolving BSA powder in phosphate- buffered solution (PBS), then filter the solution. b. Reconstitute recombinant mouse noggin, human dickkopf WNT signaling pathway inhibitor 1 (DKK-1), and Insulin-like growth factor 1 (IGF-1) to 100 pg / mE each in 0.1% BSA in PBS. Aliquot and store at -20°C. c. Reconstitute basic fibroblast growth factor (FGF-2; FGF-basic) to 10 g / mL and recombinant human / mouse / rat Activin A to 100 pg / mE each in 0.1% BSA in PBS. Aliquot and store at -80°C. d. Reconstitute SU 5402 (FGF receptor- specific tyrosine kinase inhibitor) and CHIR99021 (glycogen synthase kinase 3, GSK-3P, inhibitor) to 10 mM each in DMSO. Aliquot and store at - 20°C. Day 0 and / or Day 14 Solutions: lx Ethylenediaminetetraacetic acid (EDTA) solution (0.2 g EDTA per 1 L of PBS) lx PBS -I- (PBS without calcium or magnesium, pH 7.4). lx trypsin-like dissociation enzyme (TDE)

[0170] DPBS (Dulbecco's PBS).

[0171] RPE supporting medium (RSM)

[0172] Y-27632 dihydrochloride, Rock inhibitor (use at 10 pM).

[0173] IPSC Differentiation Protocol:

[0174] Day 0: Day of Pluripotent Stem Cell Passage for Differentiation

[0175] 1. Grow stem cell colonies in feeder- free, serum-free conditions to approximately 80% confluence before passaging.

[0176] 2. Coat a 12- well plate with extracellular matrix-based hydrogel (ECMH =Matrigel) as per manufacturer recommendations. Allow to set for 1 h at room temperature or overnight at 4 °C.

[0177] 3. Passage a single well of a 6-well plate into 4 wells of a 12- well plate (1:4).

[0178] * Aliquot the volume of RDM and PBS -I- needed for day 0 and warm in a water bath to 37 °C before adding the following growth factors: 10 mM NIC, 50 ng / mL noggin, 10 ng / mL DKK-1, and 10 ng / mL IGF-1.

[0179] Day 1 to 14: Addition of Growth Factors

[0180] 1. Day 1: Change the medium on all wells (1 mL per well) using RDM with the growth factor composition for day 0.

[0181] 2. Day 2: Change the medium using RDM (1 mL per well) with 10 mM NIC, 5 ng / mL FGF-basic, 10 ng / mL noggin, 10 ng / mL DKK-1, and 10 ng / mL IGF-1.

[0182] 3. Day 4: Change the medium using RDM (1 mL per well) with 100 ng / mL activin A, 10 ng / mL DKK-1, and 10 ng / mL IGF-1.

[0183] 4. Day 6: Change the medium using RDM (1 mL per well) with 100 ng / mL activin A and 10 pM SU 5402.

[0184] 5. Days 8, 10, and 12: Change the medium using RDM (1 mL per well) with 100 ng / mL activin A, 10 pM SU 5402, and 3 pM CHIR99021.

[0185] Day of Enrichment to Passage 0 of RPE:

[0186] Start by coating a 6-well plate with growth factor reduced ECMH (GFR-Matrigel) as per the manufacturer's guidelines and let it set. Prepare the required solutions and mediums by warming them to 37 °C. For the initial 4-7 days, use RSM mixed with Antimicrobial Reagent and Y-27632 Rock inhibitor. To make RSM mixed with Antimicrobial Reagent and Y-27632 Rock inhibitor add antimicrobial reagent and Y-27632 obtaining 0.5x and 10 |aM compositions respectively to RSM.

[0187] To prepare the cells, first, remove old media and replace with RDM. Under a microscope, remove all non-RPE cells. After clearing away debris and optional washing, add TDE to the cells and incubate. Using a cell scraper, lift the cells, and triturate to create a uniform suspension. After diluting and centrifuging the suspension, resuspend the cells in RSM with 10 pM Y-27632 (1 mL per enriched well). Then, strain the cells using a nylon mesh cell strainer with 40 pm pores and determine their concentration.

[0188] Seed the strained cells onto the growth factor reduced ECM-coated plates at 1 x 10scells / cmUn 4 mL of RSM with 10 pM Y-27632. After 48 hours, refresh the medium with RSM and 10 pM Y-27632. After a week, continue refreshing only with RSM every 3-4 days for about a month, ensuring the cells mature under the appropriate conditions.

[0189] Maturation of RPE: Passage 1 and 2 of RPE:

[0190] Between days 28 to 35 of passage 0, prepare a 6-well plate with ECMH (Matrigel).

[0191] Warm the required volume of DPBS and RSM and bring TDE (TrypLE™ Select (Thermo Fisher Scientific)) to room temperature. Remove old medium from wells, wash with DPBS, and treat with TrypLE™ Select (Thermo Fisher Scientific). Observe cell detachment under a microscope. Gently scrape and triturate cells to form a uniform suspension, then dilute in RSM. After centrifuging, resuspend the cell pellet in RSM and strain. Count and determine cell concentration, then seed onto the prepared ECMH-coated plates. Allow for a 30-day maturation period with periodic RSM changes. This process should be repeated at day 30 for passaging.

[0192] For cryopreservation, ensure cells are subconfluent and without pigment. Calculate the needed volume of cryopreservation medium with DMSO based on cell numbers. After following the passaging steps, resuspend the cells in the cryopreservation medium, and aliquot into cryogenic vials. Freeze vials at a controlled rate in a freezing container overnight at -80 °C before transferring to liquid nitrogen for long-term storage.

[0193] Sequence

[0194] The sequence used to create the inducible NGN2 cell line was from pUCM-AAVSl-TO- hNGN2 and is shown below: AAGGGCGAATTCAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACAT

[0195] TTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGG

[0196] TGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAG

[0197] CGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCT

[0198] ATGTGGCGCGGTATTATCCCGTATTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTC

[0199] AGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGA

[0200] ATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAG

[0201] GACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAA

[0202] CCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTGTAGCAATGGCAACAA

[0203] CGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATG

[0204] GAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAA

[0205] ATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCC

[0206] GTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGA

[0207] GATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGA

[0208] TTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAAT

[0209] CCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAG

[0210] ATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTT

[0211] TGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAAT

[0212] ACTGTTCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTC

[0213] GCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCA

[0214] AGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCT

[0215] TGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCC

[0216] CGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGA

[0217] G

[0218] AC TTT TT TC TC GA TG GG AG TG GG CA TA CA GC TG CC AC GT GG GG GT GA GT CC GT GT ATA GT CA CG TATC TC GT GG AT AC AG AG AG CT GT CT CC AG GC CC AA AC CC GTC CT GG GA CC CT TT TG TTA TG AC CG GT GC TG

[0219] TCCTGGCGAATTCGCCCTTAGAGCAGAGCCAGGAACCCCTGTAGGGAAGGGGCAGGAGAGCCAGGGG

[0220] CATGAGATGGTGGACGAGGAAGGGGGACAGGGAAGCCTGAGCGCCTCTCCTGGGCTTGCCAAGGACT

[0221] CAAACCCAGAAGCCCAGAGCAGGGCCTTAGGGAAGCGGGACCCTGCTCTGGGCGGAGGAATATGTCC

[0222] CAGATAGCACTGGGGACTCTTTAAGGAAAGAAGGATGGAGAAAGAGAAAGGGAGTAGAGGCGGCCA

[0223] CGACCTGGTGAACACCTAGGACGCACCATTCTCACAAAGGGAGTTTTCCACACGGACACCCCCCTCCT

[0224] CACCACAGCCCTGCCAGGACGGGGCTGGCTACTGGCCTTATCTCACAGGTAAAACTGACGCACGGAG

[0225] GAACAATATAAATTGGGGACTAGAAAGGTGAAGAGCCAAAGTTAGAACTCAGGACCAACTTATTCTG

[0226] ATTTTGTTTTTCCAAACTGCTTCTCCTCTTGGGAAGTGTAAGGAAGCTGCAGCACCAGGATCAGTGAAA

[0227] CGCACCAGACAGCCGCGTCAGAGCAGCTCAGGTTCTGGGAGAGGGTAGCGCAGGGTGGCCACTGAGA

[0228] ACCGGGCAGGTCACGCATCCCCCCCTTCCCTCCCACCCCCTGCCAAGCTCTCCCTCCCAGGATCCTCTC

[0229] TGGCTCCATCGTAAGCAAACCTTAGAGGTTCTGGCAAGGAGAGAGATGGCTCCAGGAAATGGGGGTG

[0230] TGTCACCAGATAAGGAATCTGCCTAACAGGAGGTGGGGGTTAGACCCAATATCAGGAGACTAGGAAG

[0231] GAGGAGGCCTAAGGATGGGGCTTTTCTGTCACCAATCCTGTCCCTAGTAAAGCTGATATCTCCACCCC

[0232] ACAGTGGTTTACTCCCTATCAGTGATAGAGAACGTATGAAGAGTTTACTCCCTATCAGTGATAGAGAA

[0233] CGTATGCAGACTTTACTCCCTATCAGTGATAGAGAACGTATAAGGAGTTTACTCCCTATCAGTGATAG

[0234] AGAACGTATGACCAGTTTACTCCCTATCAGTGATAGAGAACGTATCTACAGTTTACTCCCTATCAGTGA

[0235] TAGAGAACGTATATCCAGTTTACTCCCTATCAGTGATAGAGAACGTATAAGCTTTAGGCGTGTACGGT

[0236] GGGCGCCTATAAAAGCAGAGCTCGTTTAGTGAACCGTCAGATCGCCTGGAGCAATTCCACAACACTTT

[0237] TGTCTTATACCAACTTTCCGTACCACTTCCTACCCTCGTAAACTTAAGGTTAATTAAGCCGCCACCATG

[0238] TTCGTCAAATCCGAGACCTTGGAGTTGAAGGAGGAAGAGGACGTGTTAGTGCTGCTCGGATCGGCCTC

[0239] CCCCGCCTTGGCGGCCCTGACCCCGCTGTCATCCAGCGCCGACGAAGAAGAGGAGGAGGAGCCGGGC

[0240] GCGTCAGGCGGGGCGCGTCGGCAGCGCGGGGCTGAGGCCGGGCAGGGGGCGCGGGGCGGCGTGGCT

[0241] GCGGGTGCGGAGGGCTGCCGGCCCGCACGGCTGCTGGGTCTGGTACACGATTGCAAACGGCGCCCTTC

[0242] CCGGGCGCGGGCCGTCTCCCGAGGCGCCAAGACGGCCGAGACGGTGCAGCGCATCAAGAAGACCCGT

[0243] AGACTGAAGGCCAACAACCGCGAGCGAAACCGCATGCACAACCTCAACGCGGCACTGGACGCGCTGC

[0244] GCGAGGTGCTCCCCACGTTCCCCGAGGACGCCAAGCTCACCAAGATCGAGACCCTGCGCTTCGCCCAC

[0245] AACTACATCTGGGCACTCACCGAGACCCTGCGCCTGGCGGATCACTGCGGGGGCGGCGGCGGGGGCC

[0246] TGCCGGGGGCGCTCTTCTCCGAGGCAGTGTTGCTGAGCCCGGGAGGCGCCAGCGCCGCCCTGAGCAGC

[0247] AGCGGAGACAGCCCCTCGCCCGCCTCCACGTGGAGTTGCACCAACAGCCCCGCGCCGTCCTCCTCCGT

[0248] GTCCTCCAATTCCACCTCCCCCTACAGCTGCACTTTATCGCCCGCCAGCCCGGCCGGGTCAGACATGGA

[0249] CTATTGGCAGCCCCCACCTCCCGACAAGCACCGCTATGCACCTCACCTCCCCATAGCCAGGGATTGTA

[0250] TCTTGTAAGCGGCCGCAATCGATCGCCCACCCCAACTTGTTTATTGCAGCTTATAATGGTTACAAATAA

[0251] AGCAATAGCATCACAAATTTCACAAATAAAGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCAAA

[0252] CTCATCAATGTATCTTATCATGTCTGTATACCGTCGACCTCTAGCTAGAGCTTGGCGTAATCATGGTCA

[0253] TAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAA GTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTT

[0254] CCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTG

[0255] CGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCG

[0256] GTATCAGCTCACTCAAAGGACGGCCGCACATGTAAGGAAAATTTTAGGGATGTTAAAGAAAAAAATA

[0257] ACACAAAACAAAATATAAAAAAAATCTAACCTCAAGTCAAGGCTTTTCTATGGAATAAGGAATGGAC

[0258] AGCAGGGGGCTGTTTCATATACTGATGACCTCTTTATAGCCAACCTTTGTTCATGGCAGCCAGCATATG

[0259] GGCATATGTTGCCAAACTCTAAACCAAATACTCATTCTGATGTTTTAAATGATTTGCCCTCCCATATGT

[0260] CCTTCCGAGTGAGAGACACAAAAAATTCCAACACACTATTGCAATGAAAATAAATTTCCTTTATTAGC

[0261] CAGAAGTCAGATGCTCAAGGGGCTTCATGATGTCCCCATAATTTTTGGCAGAGGGAAAAAGATCTCAG

[0262] TGGTATTTGTGAGCCAGGGCATTGGCCACACCAGCCACCACCTTCTGATAGGCAGCCTGCACCTGAGG

[0263] AGTGAATTCACGCGTTTTAGCCTGGCAGCATATCGAGATCGAAGTCATCGAGAGCGTCAGCAGGGAGC

[0264] ATATCCAGATCAAAATCATCCAGGGCATCGGCGGGCAGCATGTCCAGGTCGAAGTCGTCCAGGGCGTC

[0265] GGTGGGTCCGCCGCTCTCGCACTTCAGCTGCTTCTCGAGGCCGCAGATGATCAGTTCCAGGCCGAACA

[0266] GGAAGGCGGGCTCGGCGCCCTGTCTGTCGAACAGCTCGATGGCCTGCTTCAGCAGGGGGGGCATGCTG

[0267] TCGGTGGTGGGTGTCTCTCTTTCCTCTTTGGCGACCTGGTGTTCCTGTTCTTCCAGCACGCAGCCCAGG

[0268] GTGAAGTGGCCCACGGCGCTCAGGGCGTACAGGGCGTTTTCCAGGCTGAAGCCCTGCTGGCACAGGA

[0269] AGGCCAGCTGGTTTTCCAGTGTCTCGTACTGCTTCTCGGTGGGTCTGGTGCCCAGGTGCACTTTGGCGC

[0270] CGTCCCGGTGGCTCAGCAGGGCGCATCTGTAGCTCTTGGCGTTGTTCCGCAGGAAGTCCTGCCAGCTCT

[0271] CGCCTTCCAGAGGGCAGCTGTGGGTGTGGTGCCGGTCCAGCATCTCGATGGGCAGGGCGTCCAGCAGG

[0272] GCCCGCTTGTTCTTCACGTGCCAGTACAGGGTGGGCTGTTCCACGCCCAGTTTCTGGGCCAGCTTCCGG

[0273] GTGGTCAGGCCCTCGATGCCCACGCCGTTCAGCAGTTCCAGGGCGCTGTTGATCACTTTGCTCTTGTCC

[0274] AGCCGGCTCATGTCGACATTCTTTGCCAAAATGATGAGACAGCACAATAACCAGCACGTTGCCCAGGA

[0275] GCTGTAGGAAAAAGAAGAAGGCATGAACATGGTTAGCAGAGGCTCTAGAGCCGCCGGTCACACGCCA

[0276] GAAGCCGAACCCCGCCCTGCCCCGTCCCCCCCGAAGGCAGCCGTCCCCCCGCGGACAGCCCCGAGGCT

[0277] GGAGAGGGAGAAGGGGACGGCGGCGCGGCGACGCACGAAGGCCCTCCCCGCCCATTTCCTTCCTGCC

[0278] GGCGCCGCACCGCTTCGCCCCGCGCCCGCTAGAGGGGGTGCGGCGGCGCCTCCCAGATTTCGGCTCCG

[0279] CACAGATTTGGGACAAAGGAAGTCCCTGCGCCCTCTCGCACGATTACCATAAAAGGCAATGGCTGCGG

[0280] CTCGCCGCGCCTCGACAGCCGCCGGCGCYCCGGGGGCCGCCGCGCCCCYCCCCCGAGCCCYCCCCGGC

[0281] CCGRGGCGGCCCCGCCCCGCCCGGCACCCCCMCCTGCCGCCACCCCCCGCCCGGCACGGCGAGCCCC

[0282] GCGCCACGCCCCGTACGGAGCCCCGCACCCGAAGCCGGGCCGTGCTCAGCAACTCGGGGAGGGGGGT

[0283] GCAGGGGGGGTTGCAGCCCGACCGACGCGCCCACACCCCCTGCTCACCCCCCCACGCACACACCCCGC

[0284] ACGCAGCCTTTGTTCCCCTCGCAGCCCCCCCCGCACCGCGGGGCACCGCCCCCGGCCGCGCTCCCCTC

[0285] GCGCACACTGCGGAGCGCACAAAGCCCCGCGCCGCGCCCGCAGCGCTCACAGCCGCCGGGCAGCGCG

[0286] GAGCCGCACGCGGCGCTCCCCACGCACACACACACGCACGCACCCCCCGAGCCGCTCCCCCCGCACA

[0287] AAGGGCCCTCCCGGAGCCCCTCAAGGCTTTCACGCAGCCACAGAAAAGAAACAAGCCGTCATTAAAC

[0288] CAAGCGCTAATTACAGCCCGGAGGAGAAGGGCCGTCCCGCCCGCTCACCTGTGGGAGTAACGCGGTC

[0289] AGTCAGAGCCGGGGCGGGCGGCGCGAGGCGGCGGCGGAGCGGGGCACGGGGCGAAGGCAGCGCGCA

[0290] GCGACTCCCGCCCGCCGCGCGCTTCGCTTTTTATAGGGCCGCCGCCGCCGCCGCCTCGCCATAAAAGG

[0291] AAACTTTCGGAGCGCGCCGCTCTGATTGGCTGCCSCCSCMCCYCYCCSCCYCSCCCCSCCCCSCCCCYCS

[0292] CCCCSCCCCSCCCCSCCYSGSGCGCSCCCCCCCCCCCCCCCCGCCCCCATCGCTGCACAAAATAATTAAA

[0293] AAATAAATAAATACAAAATTGGGGGTGGGGAGGGGGGGGAGATGGGGAGAGTGAAGCAGAACGTGG

[0294] GGCTCACCTCGACCATGGTAATAGCGATGACTAATACGTAGATGTACTGCCAAGTAGGAAAGTCCCAT

[0295] AAGGTCATGTACTGGGCATAATGCCAGGCGGGCCATTTACCGTCATTGACGTCAATAGGGGGCGTACT

[0296] TGGCATATGATACACTTGATGTACTGCCAAGTGGGCAGTTTACCGTAAATACTCCACCCATTGACGTC

[0297] AATGGAAAGTCCCTATTGGCGTTACTATGGGAACATACGTCATTATTGACGTCAATGGGCGGGGGTCG

[0298] TTGGGCGGTCAGCCAGGCGGGCCATTTACCGTAAGTTATGTAACGCGGAACTCCATATATGGGCTATG

[0299] AACTAATGACCCCGTAATTGATTACTATTAATAACTAGTCAATAATCAATGTCGAGACCCATAGAGCC

[0300] CACCGCATCCCCAGCATGCCTGCTATTGTCTTCCCAATCCTCCCCCTTGCTGTCCTGCCCCACCCCACCC

[0301] CCCAGAATAGAATGACACCTACTCAGACAATGCGATGCAATTTCCTCATTTTATTAGGAAAGGACAGT

[0302] GGGAGTGGCACCTTCCAGGGTCAAGGAAGGCACGGGGGAGGGGCAAACAACAGATGGCTGGCAACT

[0303] AGAAGGCACAGTCGAGGCTGATCAGCGGGTTTATAACTTCGTATAATGTATGCTATACGAAGTTATGG

[0304] TACCTTACTCGTCCATGCCGCCGGTGGAGTGGCGGCCCTCGGCGCGTTCGTACTGTTCCACGATGGTGT

[0305] AGTCCTCGTTGTGGGAGGTGATGTCCAACTTGATGTTGACGTTGTAGGCGCCGGGCAGCTGCACGGGC

[0306] TTCTTGGCCTTGTAGGTGGTCTTGACCTCAGCGTCGTAGTGGCCGCCGTCCTTCAGCTTCAGCCTCTGC

[0307] TTGATCTCGCCCTTCAGGGCGCCGTCCTCGGGGTACATCCGCTCGGAGGAGGCCTCCCAGCCCATGGT

[0308] CTTCTTCTGCATTACGGGGCCGTCGGAGGGGAAGTTGGTGCCGCGCAGCTTCACCTTGTAGATGAACT

[0309] CGCCGTCCTGCAGGGAGGAGTCCTGGGTCACGGTCACCACGCCGCCGTCCTCGAAGTTCATCACGCGC

[0310] TCCCACTTGAAGCCCTCGGGGAAGGACAGCTTCAAGTAGTCGGGGATGTCGGCGGGGTGCTTCACGTA

[0311] GGCCTTGGAGCCGTACATGAACTGAGGGGACAGGATGTCCCAGGCGAAGGGCAGGGGGCCACCCTTG

[0312] GTCACCTTCAGCTTGGCGGTCTGGGTGCCCTCGTAGGGGCGGCCCTCGCCCTCGCCCTCGATCTCGAAC TCGTGGCCGTTCACGGAGCCCTCCATGTGCACCTTGAAGCGCATGAACTCCTTGATGATGGCCATGTTA

[0313] TCCTCCTCGCCCTTGCTCACCATGGTGGCGACCGGTAGCGCTAGCGTAGGCGCCGGTCACAGCTTGGA

[0314] TCTGTAACGGCGCAGAACAGAAAACGAAACAAAGACGTAGAGTTGAGCAAGCAGGGTCAGGCAAAG

[0315] CGTGGAGAGCCGGCTGAGTCTAGGTAGGCTCCAAGGGAGCGCCGGACAAAGGCCCGGTCTCGACCTG

[0316] AGCTTTAAACTTACCTAGACGGCGGACGCAGTTCAGGAGGCACCACAGGCGGGAGGCGGCAGAACGC

[0317] GACTCAACCGGCGTGGATGGCGGCCTCAGGTAGGGCGGCGGGCGCGTGAAGGAGAGATGCGAGCCCC

[0318] TCGAAGCTTCAGCTGTGTTCTGGCGGCAAACCCGTTGCGAAAAAGAACGTTCACGGCGACTACTGCAC

[0319] TTATATACGGTTCTCCCCCACCCTCGGGAAAAAGGCGGAGCCAGTACACGACATCACTTTCCCAGTTT

[0320] ACCCCGCGCCACCTTCTCTAGGCACCGGTTCAATTGCCGACCCCTCCCCCCAACTTCTCGGGGACTGTG

[0321] GGCGATGTGCGCTCTGCCCACTGACGGGCACCGGAGCGATCGCAGATCATGCATCCATAGAGCCCACC

[0322] GCATCCCCAGCATGCCTGCTATTGTCTTCCCAATCCTCCCCCTTGCTGTCCTGCCCCACCCCACCCCCCA

[0323] GAATAGAATGACACCTACTCAGACAATGCGATGCAATTTCCTCATTTTATTAGGAAAGGACAGTGGGA

[0324] GTGGCACCTTCCAGGGTCAAGGAAGGCACGGGGGAGGGGCAAACAACAGATGGCTGGCAACTAGAA

[0325] GGCACAGGTTTAAACGGGCCCTCTAGATCAGGCACCGGGCTTGCGGGTCATGCACCAGGTGCGCGGTC

[0326] CTTCGGGCACCTCGACGTCGGCGGTGACGGTGAAGCCGAGCCGCTCGTAGAAGGGGAGGTTGCGGGG

[0327] CGCGGAGGTCTCCAGGAAGGCGGGCACCCCGGCGCGCTCGGCCGCCTCCACTCCGGGGAGCACGACG

[0328] GCGCTGCCCAGACCCTTGCCCTGGTGGTCGGGCGAGACGCCGACGGTGGCCAGGAACCACGCGGGCT

[0329] CCTTGGGCCGGTGCGGCGCCAGGAGGCCTTCCATCTGTTGCTGCGCGGCCAGCCGGGAACCGCTCAAC

[0330] TCGGCCATGCGCGGGCCGATCTCGGCGAACACCGCCCCCGCTTCGACGCTCTCCGGCGTGGTCCAGAC

[0331] CGCCACCGCGGCGCCGTCGTCCGCGACCCACACCTTGCCGATGTCGAGCCCGACGCGCGTGAGGAAG

[0332] AGTTCTTGCAGCTCGGTGACCCGCTCGATGTGGCGGTCCGGATCGACGGTGTGGCGCGTGGCGGGGTA

[0333] GTCGGCGAACGCGGCGGCGAGGGTGCGTACGGCCCTGGGGACGTCGTCGCGGGTGGCGAGGCGCACC

[0334] GTGGGCTTGTACTCGGTCATCTCGAGCCTAGGGCCGGGATTCTCCTCCACGTCACCGCATGTTAGAAG

[0335] ACTTCCTCTGCCCTCTCCGCTGCCAGATCTCTCGAGGCCCTGTGGGAGGAAGAGAAGAGGTCAGAAGC

[0336] TTATAACTTCGTATAATGTATGCTATACGAAGTTATTGCCCCACTGTGGGGTGGAGGGGACAGATAAA

[0337] AGTACCCAGAACCAGAGCCACATTAACCGGCCCTGGGAATATAAGGTGGTCCCAGCTCGGGGACACA

[0338] GGATCCCTGGAGGCAGCAAACATGCTGTCCTGAAGTGGACATAGGGGCCCGGGTTGGAGGAAGAAGA

[0339] CTAGCTGAGCTCTCGGACCCCTGGAAGATGCCATGACAGGGGGCTGGAAGAGCTAGCACAGACTAGA

[0340] GAGGTAAGGGGGGTAGGGGAGCTGCCCAAATGAAAGGAGTGAGAGGTGACCCGAATCCACAGGAGA

[0341] ACGGGGTGTCCAGGCAAAGAAAGCAAGAGGATGGAGAGGTGGCTAAAGCCAGGGAGACGGGGTACT

[0342] TTGGGGTTGTCCAGAAAAACGGTGATGATGCAGGCCTACAAGAAGGGGAGGCGGGACGCAAGGGAG

[0343] ACATCCGTCGGAGAAGGCCATCCTAAGAAACGAGAGATGGCACAGGCCCCAGAAGGAGAAGGAAAA

[0344] GGGAACCCAGCGAGTGAAGACGGCATGGGGTTGGGTGAGGGAGGAGAGATGCCCGGAGAGGACCCA

[0345] GACACGGGGAGGATCCGCTCAGAGGACATCACGTGGTGCAGCGCCGAGAAGGAAGTGCTCCGGAAAG

[0346] AGCATCCTTGGGCAGCAACACAGCAGAGAGCAAGGGGAAGAGGGAGTGGAGGAAGACGGAACCTGA

[0347] AGGAGGCGGCAGGGAAGGATCTGGGCCAGCCGTAGAGGTGACCCAGGCCACAAGCTGCAGACAGAA

[0348] AGCGGCACAGGCCCAGGGGAGAGAATGCTGGTCAGAGAAAGCA (SEQ ID NO: 2)

[0349] Example 2. Optimal cellular target / timing of differentiation for reprogramming of RPE fate to neural retina.

[0350] To generate photoreceptors from PSC-derived RPE the following study was performed to identify (1) the optimal stage for inducing expression of the master regulatory transcription factor, and (2) the purification of cells at the optimal stage of differentiation. Without wishing to be bound by theory, induction of master regulatory transcription factor expression may occur at any time during the RPE differentiation (FIG. 1). However, the outcome is dependent on the differentiated state of the culture at a given stage. Induction of NGN2 expression in PSC (Day 0) resulted in the generation of cortical neurons (FIG. 3). These results suggest that if a preparation to be reprogrammed contains residual pluripotent stem cells, then after inducing expression of the master regulatory transcription factor, the cellular product might become contaminated with cortical neurons. Induction of NGN2 was delayed until approximately 70 days of RPE differentiation, when the RPE cells were at full maturation. The results show that in the RPE cells induced at 70 days of differentiation, expression of photoreceptor markers was observed, although the efficiency is low (FIG. 4). Higher efficiencies of expression of photoreceptor markers were obtained in intermediate stages (FIGs. 2A-2C).

[0351] To determine the optimal stage for inducing expression of the master regulatory transcription factor, expression of candidate stage specific differentiation markers likely to be expressed during the course of RPE differentiation and maturation was examined.

[0352] The timing of marker expression during the phase of differentiation of RPE (DO- 14, FIG. 1) was examined. The results show that OTX2, a general marker for early neural precursors, was turned off by D4 of the differentiation protocol (FIG. 5A). PAX6, a maker for neural and eye progenitors, was detected by D4 and was widely expressed thereafter (FIG. 5B). ZO1, a marker for early stages of RPE differentiation, was expressed from Day 1 onwards, but only assumed its typical epithelial appearance at the borders of the cell at DIO- 12 (FIG. 5A). MITF, a marker of RPE lineage, appeared at DIO- 12 (FIG. 5B). NCAM was found strongly expressed on the cell surface on D12 (FIG. 5C). KI67, a marker for proliferation, was expressed in most cells up to D8, when it began to decline (FIG. 5D). These results show that NCAM positive cells are candidate stage specific progenitor for induction of reprogramming. NCAM is expressed on the cell surface near the end of the differentiation phase, prior to maturation. When cell surface NCAM appeared on most of the population, ZO1 is similarly expressed on the surface, MITF is induced, OTX2 is downregulated, and the percent of KI67 positive cells declined considerably. Thus, NCAM positive cells have undergone differentiation and are preparing to enter maturation phase. The finding that KI67 staining declined as NCAM is activated allows for the identification of the presence of proliferating cells in a graft, which might compromise safety.

[0353] Using the protocol described herein, NCAM positive cells or cells at a similar stage expressing other cell surface markers can be isolated by various techniques including fluorescence activated cell sorting or magnetic based cell sorting, and replated for immediate reprogramming, or for further maturation as RPE, for use as unmodified RPE in grafts or for later reprogramming. Purification is performed because cultures can remain heterogeneous even after 12 days of differentiation (FIG. 5E); a small minority of remaining OTX2+ZO1- cells illustrated here could yield unwanted diverse outcomes during reprogramming. Example 3. Construction of a graft for generating RPE and photoreceptors.

[0354] The combination of differentiated RPE and RPE capable of being reprogrammed to a neural retina fate within the same construct provides for the replacement of diseased RPE and regeneration of photoreceptors. To replace diseased RPE and regenerate photoreceptors, grafts were constructed consisting of mature RPE and an RPE progenitor cell capable of efficient reprogramming into the neural retinal lineage following activation of an engineered master regulatory transcription factor, such as NGN2. The mature RPE expressed relevant markers of maturation including BEST1, RPE65, PEDF, F0LR1, and others known in the art and indices of functional maturation such as levels of transepithelial membrane resistance >100 / cm2, and appropriate structural features (apical microvilli, tight junctions, melanosomes, polarization).

[0355] A population consisting of inappropriate amounts of either mature RPE or reprogrammable RPE or precursor cells could fail to produce adequate numbers of RPE or photoreceptors to restore and maintain vision in the patient. For example, in FIGs. 2A-2C most cells in the culture, produced from a population consisting solely of cells bearing an inducible NGN2 construct, have almost all begun to express markers of the photoreceptor lineage; mature RPE markers have mainly disappeared. This is also illustrated in FIG. 6, which shows that the epithelial cells have disappeared altogether after NGN2 induction; this dissolution of RPE morphology is also seen during regeneration of neural retina from RPE in the newt (Islam et al., 2014).

[0356] After reprogramming in a monolayer culture, the photoreceptors were arrayed in a monolayer with no particular structural organization (FIGs. 2A-2C). Grafts were designed to yield the anatomical arrangement depicted in FIG. 7, with a layer of RPE underlying the newly generated photoreceptors.

[0357] Combining a population of wild type RPE with RPE having an inducible NGN2 construct in a 50:50 ratio, plating the cells, and then inducing NGN2 expression, yielded a monolayer with a patchwork of cells having photoreceptor and RPE markers. (FIGs. 8A-8B). Next, a cellular bilayer of wild type RPE and NGN2 inducible cells were generated using the following protocol:

[0358] Transdifferentiation of Human PSC-Derived RPE Cells into Photoreceptors by Overexpression of an Inducible Ngn2 in a Bilayer System

[0359] 1. Plate WT PSC-derived RPE cells derived from PSC.

[0360] 2. NGN2-inducible RPE are to be differentiated as described in Examples 1 and 2.

[0361] 3. Subculture NGN2-inducible RPE cell line 4. Aspirate spent media.

[0362] 5. Wash with room temperature PBS once.

[0363] 6. To a 100 mm dish, add 5 ml of TrypLE™ Select (Thermo Fisher Scientific).

[0364] 7. Incubate at 37 °C for 5 to 10 minutes. During this time, take the plate out of the incubator a couple of times and tap it to accelerate the dislodgement of the cells.

[0365] 8. After that time, cells will not be completely detached, use a cell scraper to lift the cells from the plasticware surface.

[0366] 9. Using a P1000, pipette up and down up to 5 times to further dissociate the cells.

[0367] 10. Collect the samples and transfer them to a 15 ml conical tube, rinse the dish with fresh complete media to recover the remaining cells. Transfer the cells to the same tube and inactivate TrypLE™ Select (Thermo Fisher Scientific) with 1: 1 suspension of complete media to TrypLE™ Select (Thermo Fisher Scientific). Do not let the enzyme be active for more than 20 min.

[0368] 11. Centrifuge the cells at 250g for 5 min and aspirate the media.

[0369] 12. Resuspend cells in Retinal differentiation media (RDM) containing Ipg / ml Doxycycline and 10 pM of Y-27632.

[0370] 13. Seed the cells in the same area they came from but on a plasticware containing the aforementioned WT PSC-derived RPE cells.

[0371] 14. Next day remove media and replace it with fresh RDM plus doxycycline.

[0372] 15. Media should be changed every other day with doxycycline for 2 weeks.

[0373] 16. Continue to grow the cells for one more week without doxycycline.

[0374] At this point cells will be expressing photoreceptor markers Retinal differentiation media

[0375] • DMEM / F- 12 (Thermo 31330095)

[0376] • 1% (v / v) nonessential amino acids (Thermo 11140068)

[0377] • 1% (v / v) penicillin-streptomycin (Thermo 15140122)

[0378] • 2% (v / v) B-27 supplement (Thermo 17504001)

[0379] • 1% (v / v) N-2 supplement (Thermo 17502001)

[0380] • 10% (v / v) Fetal Bovine Serum (Thermo A2720801) o mM Taurine (Sigma-Aldrich T8691)

[0381] 40 ng / ml T3 (Sigma-Aldrich T6397)

[0382] 10 ng / ml IGF-1 (Sigma- Aldrich SRP3069)

[0383] Add 0.5 pM retinoic acid immediately before use (Sigma- Aldrich SRP3069) This bilayer culture protocol yielded a monolayer of RPE that continued to express markers of mature RPE along with large foci of cells that expressed markers of neural retina above the plane of the RPE monolayer (FIGs. 9A-9B). These cultures were generated on tissue culture plastic, but for enhanced maturation and further differentiation, they can be grown on a system that allows for polarization of RPE, and further differentiation and maturation of RPE and neural retina, for prolonged period. This might include culture on permeable transwell filters or permeable substrates, such as might be suitable for subsequent engraftment into experimental animals or patients.

[0384] ADDITIONAL EMBODIMENTS

[0385] All of the features disclosed in this specification may be combined in any combination. Each feature disclosed in this specification may be replaced by an alternative feature serving the same, equivalent, or similar purpose. Thus, unless expressly stated otherwise, each feature disclosed is only an example of a generic series of equivalent or similar features.

[0386] From the above description, one skilled in the art can easily ascertain the essential characteristics of the present disclosure, and without departing from the spirit and scope thereof, can make various changes and modifications of the disclosure to adapt it to various usages and conditions. Thus, other embodiments are also within the claims. For example, the Additional Embodiments below are expressly contemplated:

[0387] Additional Embodiments

[0388] Additional embodiments are encompassed by the following numbered paragraphs.

[0389] 1. A mammalian retinal pigment epithelial cell comprising an engineered activatable polynucleotide encoding a master regulatory transcription factor.

[0390] 2. The mammalian retinal pigment epithelial cell of paragraph 1, wherein the engineered activatable polynucleotide comprises an inducible promoter operably linked to an open reading frame encoding the master regulatory transcription factor.

[0391] 3. The mammalian retinal pigment epithelial cell of paragraph 1 or 2, wherein the master regulatory transcription factor is selected from Neurogenin-1 (NGN1), NGN2, NGN3, Paired Box 6 (PAX6), Neurogenic Differentiation 1 (NEURODI), SIX homeobox 2 (SIX2), and orthodenticle homeobox 2 (0TX2).

[0392] 4. The mammalian retinal pigment epithelial cell of paragraph 3, wherein the master regulatory transcription factor is NGN2. 5. The mammalian retinal pigment epithelial cell of any one of the preceding paragraphs, wherein the mammalian retinal pigment epithelial cell is an early retinal pigment epithelial cell, optionally expressing MITF, SERPINF1, and / or PMEL, or a mature retinal pigment epithelial cell, optionally expressing RPE 65, BEST1, FOLR1, SLC16A8, PDGFRA.

[0393] 6. The mammalian retinal pigment epithelial cell of any one of the preceding paragraphs, wherein the cell is derived from an induced pluripotent stem cell.

[0394] 7. The mammalian retinal pigment epithelial cell of any one of the preceding paragraphs, wherein the cell is a human cell.

[0395] 8. A subpopulation of cells, wherein each cell of the subpopulation is the mammalian retinal pigment epithelial cell of any one of the preceding paragraphs.

[0396] 9. A population of cells comprising the subpopulation of cells of paragraph 8.

[0397] 10. The population of cells of paragraph 9 further comprising retinal cells selected from photoreceptor cells, retinal ganglion cells, and Muller glial cells.

[0398] 11. A population of cells differentiated from the subpopulation of cells of paragraph 8.

[0399] 12. The population of cells of any one of paragraphs 9-11, wherein cells of the population express (a) CRX, CHX10, S-OPSIN, RHODOPSIN, BRN3, RCVRN, and / or MAP2; or (b) RAX, PAX6, CHX10, SIX3, NCAM1, and / or OTX2.

[0400] 13. A composition comprising the population of cells of any one of paragraphs 9-12.

[0401] 14. The composition of paragraph 13, wherein the composition comprises cell culture media.

[0402] 15. A spheroid comprising the population of cells of any one of paragraphs 9-12.

[0403] 16. An organoid comprising the population of cells of any one of paragraphs 9-12, optionally wherein the organoid is attached to a substrate suitable for transplantation into a human eye.

[0404] 17. A cellular therapeutic device comprising the subpopulation or population of cells, the composition, the spheroid or the organoid of any one of the preceding paragraphs and optionally additional mammalian retinal pigment epithelial cells, preferably derived from pluripotent stem cells, such as induced pluripotent stem cells or embryonic stem cells.

[0405] 18. A mammalian pluripotent stem cell comprising an engineered activatable polynucleotide encoding a master regulatory transcription factor.

[0406] 19. The mammalian pluripotent stem cell of paragraph 18, wherein the cell is a human cell.

[0407] 20. The mammalian pluripotent stem cell of paragraph 18 or 19, wherein the cell is an induced pluripotent stem cells or an embryonic stem cell. 22. The mammalian pluripotent stem cell of any one of paragraphs 18-20, wherein the engineered activatable polynucleotide comprises an inducible promoter operably linked to an open reading frame encoding the master regulatory transcription factor.

[0408] 23. The mammalian pluripotent stem cell of any one of paragraphs 18-22, wherein the master regulatory transcription factor is selected from Neurogenin-1 (NGN1), NGN2, NGN3, Paired Box 6 (PAX6), Neurogenic Differentiation 1 (NEURODI), SIX homeobox 2 (SIX2), and orthodenticle homeobox 2 (OTX2).

[0409] 24. The mammalian pluripotent stem cell of paragraph 23, wherein the master regulatory transcription factor is NGN2.

[0410] 25. A method, comprising delivering to a mammalian retinal pigment epithelial cell an engineered activatable polynucleotide encoding a master regulatory transcription factor.

[0411] 26. A method, comprising delivering to a mammalian pluripotent stem cell an engineered activatable polynucleotide encoding a master regulatory transcription factor.

[0412] 27. The method of paragraph 25 or 26, wherein the engineered activatable polynucleotide comprises an inducible promoter operably linked to an open reading frame encoding a master regulatory transcription factor.

[0413] 28. A method comprising: (a) delivering to pluripotent stem cells a polynucleotide comprising an inducible promoter operably linked to an open reading frame encoding a master regulatory transcription factor and (b) culturing the pluripotent stem cells in differentiation media to produce mammalian retinal pigment epithelial cells.

[0414] 29. A method comprising: (a) culturing pluripotent stem cells in differentiation media to produce mammalian retinal pigment epithelial cells; and (b) delivering to the mammalian retinal pigment epithelial cells a polynucleotide comprising an inducible promoter operably linked to an open reading frame encoding a master regulatory transcription factor.

[0415] 30. The method of any one of paragraphs 25-29, further comprising culturing the mammalian retinal pigment epithelial cells in cell culture media, optionally in a three-dimensional culture, preferably comprising a scaffold, to produce a tissue sheet, spheroid, or an organoid, preferably wherein the three-dimensional culture comprises a substrate suitable for transplantation into a human eye.

[0416] 31. The method of paragraph 30, wherein the mammalian retinal pigment epithelial cells are cultured in cell culture media for about 15 to 25, preferably about 20 days.

[0417] 32. The method of paragraph 30 or 31, wherein the cell culture media comprises one or more growth factors selected from NIC, Noggin, DKK1, IGF-1, Activin A, SU 5402, and CHIR99021. 33. The method of any one of paragraphs 25-32 further comprising activating expression of the master regulatory transcription factor, optionally producing cells expressing (a) CRX, CHX10, S-OPSIN, RHODOPSIN, BRN3, RCVRN, and / or MAP2; or (b) RAX, PAX6, CHX10, SIX3, NCAM1, and / or 0TX2.

[0418] 34. The method of paragraph 33, wherein the activating comprises culturing the cells in the presence of an inducing agent, preferably an inducing agent that activates expression of the open reading frame.

[0419] 35. The method of any one of paragraphs 25-34, wherein the master regulatory transcription factor is selected from NGN1, NGN2, NGN3, PAX6, NEURODI, SIX3, and 0TX2.

[0420] 36. The method of paragraph 35, wherein the master regulatory transcription factor is NGN2.

[0421] 37. The method of any one of paragraphs 25-35, wherein the mammalian retinal pigment epithelial cell is an early retinal pigment epithelial cell, optionally expressing MITF, SERPINF1, and / or PMEL, or a mature retinal pigment epithelial cell, optionally expressing RPE 65, BEST1, FOLR1, SLC16A8, PDGFRA.

[0422] 38. The method of any one of paragraphs 25-37, wherein the mammalian retinal pigment epithelial cell is derived from an induced pluripotent stem cell or human embryonic stem cell

[0423] 39. The method of any one of paragraphs 25-38, wherein the mammalian retinal pigment epithelial cell is a human cell.

[0424] 40. A method comprising administering to an eye of a subject the mammalian retinal pigment epithelial cell of any one of the preceding paragraphs, the subpopulation of cells of any one of the preceding paragraphs, the population of cells of any one of the preceding paragraphs, the composition of any one of the preceding paragraphs, or the organoid of any one of the preceding paragraphs.

[0425] 41. A method comprising administering to retinal pigment epithelial cells of a subject an engineered activatable polynucleotide encoding a master regulatory transcription factor.

[0426] 42. The method of paragraph 41, wherein the engineered activatable polynucleotide comprises an inducible promoter operably linked to an open reading frame encoding a master regulatory transcription factor.

[0427] 43. The method of any one of paragraphs 41-42, further comprising inducing expression of the master regulatory transcription factor in the eye of the subject.

[0428] 44. The method of any one of paragraphs 41-43, wherein the subject has a degenerative eye disease. 45. The method of paragraph 44, wherein the degenerative eye disease is age-related macular degeneration.

[0429] 46. The method of any one of paragraphs 41-45, wherein the master regulatory transcription factor is selected from NGN1, NGN2, NGN3, PAX6, NEURODI, SIX3, and 0TX2.

[0430] 47. The method of paragraph 46, wherein the master regulatory transcription factor is NGN2.

[0431] EQUIVALENTS AND SCOPE

[0432] While several inventive embodiments have been described and illustrated herein, those of ordinary skill in the art will readily envision a variety of other means and / or structures for performing the function and / or obtaining the results and / or one or more of the advantages described herein, and each of such variations and / or modifications is deemed to be within the scope of the inventive embodiments described herein. More generally, those skilled in the art will readily appreciate that all parameters, dimensions, materials, and configurations described herein are meant to be exemplary and that the actual parameters, dimensions, materials, and / or configurations will depend upon the specific application or applications for which the inventive teachings is / are used. Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation, many equivalents to the specific inventive embodiments described herein. It is, therefore, to be understood that the foregoing embodiments are presented by way of example only and that, within the scope of the appended claims and equivalents thereto, inventive embodiments may be practiced otherwise than as specifically described and claimed. Inventive embodiments of the present disclosure are directed to each individual feature, system, article, material, kit, and / or method described herein. In addition, any combination of two or more such features, systems, articles, materials, kits, and / or methods, if such features, systems, articles, materials, kits, and / or methods are not mutually inconsistent, is included within the inventive scope of the present disclosure.

[0433] The indefinite articles “a” and “an,” as used herein in the specification and in the claims, unless clearly indicated to the contrary, should be understood to mean “at least one.”

[0434] It should also be understood that, unless clearly indicated to the contrary, in any methods claimed herein that include more than one step or act, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are recited.

[0435] In the claims, as well as in the specification above, all transitional phrases such as “comprising,” “including,” “carrying,” “having,” “containing,” “involving,” “holding,” “composed of,” and the like are to be understood to be open-ended, i.e., to mean including but not limited to. Only the transitional phrases “consisting of’ and “consisting essentially of’ shall be closed or semi-closed transitional phrases, respectively, as set forth in the United States Patent Office Manual of Patent Examining Procedures, Section 2111.03.

[0436] The terms “about” and “substantially” preceding a numerical value mean ±10% of the recited numerical value.

[0437] Where a range of values is provided, each value between and including the upper and lower ends of the range are specifically contemplated and described herein.

Claims

CLAIMSWhat is claimed is:

1. A retinal graft comprising: pluripotent stem cell (PSC)-derived retinal pigment epithelial cells that comprise an engineered polynucleotide comprising an inducible promoter operably linked to a neurogenin-2 coding sequence; and mature-derived retinal pigment epithelial cells.

2. The retinal graft of claim 1 further comprising at least one substrate selected from poly(lactic-co-glycolic acid, polycaprolactone, collagen, fibrin, hyaluronic acid, decellularized matrices, hydrogels.

3. The retinal graft of claim 1 or 2, wherein the PSC-derived retinal pigment epithelial cells are human cells.

4. The retinal graft of any preceding claim, wherein the PSC-derived retinal pigment epithelial cells have been cultured for no more than 10-14 days.

5. The retinal graft of any preceding claim, wherein the PSC-derived retinal pigment epithelial cells express neural cell adhesion molecule 1 (NCAM1).

6. The retinal graft of claim 5, wherein the PSC-derived retinal pigment epithelial cells express melanocyte inducing transcription factor (MITF) and tight junction protein 1 (ZO1).

7. The retinal graft of any preceding claim, wherein the mature-derived retinal pigment epithelial cells have been cultured for at least 21 days.

8. The retinal graft of any preceding claim, wherein the proportion of PSC-derived retinal pigment epithelial cells is about 30% to about 70%, optionally about 50%.

9. The retinal graft of any preceding claim, wherein the inducible promoter is a chemicalinducible promoter.

10. The retinal graft of claim 9, wherein the chemical-inducible promoter is a tetracyclineinducible promoter.11 The retinal graft of any preceding claim, wherein the retinal graft has been treated with an inducing agent that activates the inducible promoter.

12. A method of treating an eye disorder in a subject, the method comprising administering the retinal graft of any preceding claim to an eye of a subject in need thereof.

13. The method of claim 12, further comprising administering to the subject an inducing agent that activates the inducible promoter.

14. The method of claim 13, wherein the inducible promoter is a tetracycline-inducible promoter, and the inducing agent is doxycycline.

15. The method of any one of claims 12-14, wherein the eye disorder is characterized by loss of photoreceptors.

16. The method of claim 15, wherein the eye disorder is age-related macular degeneration.

17. A method of producing a retinal graft, the method comprising: delivering, to induced pluripotent stem cells (iPSCs), a polynucleotide comprising an inducible promoter operably linked to a neurogenin-2 coding sequence; and culturing the iPSCs in differentiation media for about 10-14 days to produce the iPSC- derived retinal pigment epithelial cells of the retinal graft of any one of claims 1-11.

18. The method of claim 17, further comprising producing a retinal graft by combining the iPSC-derived retinal pigment epithelial cells with mature-derived retinal pigment epithelial cells.

19. The method of claim 17 or 18, further comprising producing a retinal graft by combining the iPSC-derived retinal pigment epithelial cells with at least one substrate selected from poly(lactic-co-glycolic acid, polycaprolactone, collagen, fibrin, hyaluronic acid, decellularized matrices, hydrogels.

20. The method of any one or claims 17-19 further comprising treating the retinal graft with an inducing agent that activates the inducible promoter.

21. The method of claim 19 or 20, further comprising administering the retinal graft to an eye of a subject having an eye disorder characterized by loss of photoreceptors.

22. The method of claim 21, wherein the eye disorder is characterized by loss of photoreceptors.

23. The method of any one of claims 17, 21, or 22 further comprising administering to the subject an inducing agent that activates the inducible promoter.

24. The method of claim 20 or 23, wherein the inducible promoter is a tetracycline-inducible promoter, and the inducing agent is doxycycline.

25. A retinal pigment epithelial (RPE) cell comprising a polynucleotide having an inducible promoter operably linked to an open reading frame encoding a master regulatory transcription factor.

26. The RPE cell of claim 25, wherein the master regulatory transcription factor is selected from the group consisting of neurogenin 1 (NGN1), neurogenin 2 (NGN2), neurogenin 3 (NGN3), paired box 6 (PAX6), neuronal differentiation 1 (NEURODI), SIX homeobox 2 (SIX2), and orthodenticle homeobox 2 (OTX2).

27. The RPE cell of claim 25 or 26, wherein the master regulatory transcription factor is NGN2.

28. The RPE cell of any one of claims 25-27, wherein the RPE cell is differentiated from a pluripotent stem cell.

29. The RPE cell of claim 28, wherein the pluripotent stem cell is a mammalian cell.

30. The RPE cell of claim 28 or 29, wherein the pluripotent stem cell is a human cell.

31. The RPE cell of any one of claims 28-30, wherein the pluripotent stem cell is a human induced pluripotent stem cell.

32. The RPE cell of any one of claims 25-31, wherein the RPE cell expresses one or more markers selected from the group consisting of neural cell adhesion molecule 1 (NCAM1), folate receptor alpha (FOLR1), solute carrier family 16 member 8 (SLC16A8), and platelet derived growth factor receptor alpha (PDGFRA), melanocyte inducing transcription factor (MITF), and tight junction protein 1 (ZO1).

33. The RPE cell of any one of claims 25-32, wherein the RPE cell expresses NCAM1.

34. The RPE cell of any one of claims 25-33, wherein the inducible promoter is a tetracycline-inducible promoter.

35. A composition comprising the RPE cell of any one of claims 25-34.

36. A method of treating a retinal disease comprising administering to a subject having a retinal disease the RPE cell of any one of claims 25-34 and an inducing agent.

37. A bilayer culture comprising, a first population of retinal pigment epithelial (RPE) cells and a second population of RPE cells, wherein the first population of the RPE cells comprises mature-derived (e.g., wild-type) RPE cells and the second population of RPE cells comprises a polynucleotide having an inducible promoter operably linked to an open reading frame encoding a master regulatory transcription factor.

38. The bilayer culture of claim 37, wherein the first population of RPE cells and the second population of RPE cells are in a ratio of 50:50.

39. A method of treating a retinal disease comprising, administering to a subject having a retinal disease the bilayer culture of claim 37 or 38 and a substrate suitable for transplantation into an eye.

40. The method of claim 39, further comprising administering an inducing agent.

41. The method of claim 40, wherein the inducing agent is doxycycline.

42. A method of differentiating a retinal pigment epithelial (RPE) cell comprising,delivering to a pluripotent stem cell a polynucleotide having an inducible promoter operably linked to an open reading frame encoding a master regulatory transcription factor; and culturing the pluripotent stem cell to produce the RPE cell.

43. The method of claim 42, wherein the pluripotent stem cell is cultured for 10-70 days.

44. The method of claim 42 or 43, wherein the pluripotent stem cell is cultured in a three- dimensional culture.

45. The method of claim 44, wherein the three-dimensional culture comprises a scaffold, to produce a tissue sheet, spheroid, or an organoid.

46. The method of any one of claims 42-45, wherein the RPE cell is purified by flow cytometry or magnetic bead sorting.

Images