WO2019099552A1 - Procédés de génération d'épithélium pigmentaire rétinien (epr) - Google Patents

Procédés de génération d'épithélium pigmentaire rétinien (epr) Download PDF

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WO2019099552A1
WO2019099552A1 PCT/US2018/061100 US2018061100W WO2019099552A1 WO 2019099552 A1 WO2019099552 A1 WO 2019099552A1 US 2018061100 W US2018061100 W US 2018061100W WO 2019099552 A1 WO2019099552 A1 WO 2019099552A1
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polypeptide
ipscs
nucleotide sequence
pax6
mitf
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Ziqi LIU
Bruce R. Conklin
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The J. David Gladstone Institutes
The Regents Of The University Of California
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Definitions

  • the naturally occurring retinal pigment epithelium is a polarized monolayer of densely packed hexagonal cells in the mammalian eye that separates the neural retina from the choroid.
  • the cells in the RPE contain pigment granules and perform an important role in retinal physiology by forming a blood-retinal barrier and closely interacting with photoreceptors to maintain visual function by absorbing the light energy focused by the lens on the retina.
  • These cells also transport ions, water, and metabolic end products from the subretinal space to the blood and take up nutrients such as glucose, retinol, and fatty acids from the blood and deliver these nutrients to photoreceptors.
  • ophthalmic diseases such as (age-related) macular degeneration, macular dystrophies such as Stargardf s and Stargardfs-like disease, Best disease (vitelliform macular dystrophy), and adult vitelliform dystrophy or subtypes of retinitis pigmentosa, are associated with a degeneration or deterioration of the retina itself or of the RPE.
  • RPE may be cultivated in vitro.
  • the present disclosure provides methods of generating retinal pigment epithelium (RPE) from induced pluripotent stem cells (iPSCs).
  • RPE retinal pigment epithelium
  • iPSCs induced pluripotent stem cells
  • the present disclosure also provides RPE generated using a subject method as well as methods of using RPE generated according to the disclosed methods. Also provided are recombinant vectors useful for the generation of RPE according to the disclosed methods.
  • the present disclosure provides a method of generating retinal
  • the method includes introducing into the iPSCs an exogenous nucleic acid including a nucleotide sequence encoding an OTX2 polypeptide, an exogenous nucleic acid including a nucleotide sequence encoding a PAX6 polypeptide, and an exogenous nucleic acid including a nucleotide sequence encoding an MITF polypeptide to provide genetically modified iPSCs.
  • the genetically modified iPSCs are cultured under conditions sufficient for expression of the OTX2 polypeptide, the PAX6 polypeptide, and the MITF polypeptide in the genetically modified iPSCs, and differentiation of the genetically modified iPSCs into retinal pigment epithelial cells.
  • the method does not include introducing into the iPSCs or genetically modified iPSCs a transcription factor other than an OTX2 polypeptide, a PAX6 polypeptide, and/or an MITF polypeptide, or an exogenous nucleic acid including a nucleotide sequence encoding a transcription factor other than an OTX2 polypeptide, a PAX6 polypeptide, and/or an MITF polypeptide. In this manner, RPE is generated.
  • the present disclosure provides a method of generating RPE from iPSCs, wherein the method includes introducing into the iPSCs an exogenous nucleic acid including a nucleotide sequence encoding an OTX2 polypeptide, an exogenous nucleic acid including a nucleotide sequence encoding a PAX6 polypeptide, and an exogenous nucleic acid including a nucleotide sequence encoding an MITF polypeptide to provide genetically modified iPSCs.
  • the genetically modified iPSCs are cultured under conditions sufficient for expression of the OTX2 polypeptide, the PAX6 polypeptide, and the MITF polypeptide in the genetically modified iPSCs, and differentiation of the genetically modified iPSCs into retinal pigment epithelial cells.
  • the method does not include introducing into the iPSCs or genetically modified iPSCs an exogenous nucleic acid including a tyrosinase enhancer. In this manner, RPE is generated.
  • the present disclosure provides a method of generating RPE from iPSCs, wherein the method includes introducing into the iPSCs a recombinant vector including an inducible promoter, e.g., a Tet-inducible promoter, an exogenous nucleic acid including a nucleotide sequence encoding an OTX2 polypeptide, an exogenous nucleic acid including a nucleotide sequence encoding a PAX6 polypeptide, and an exogenous nucleic acid including a nucleotide sequence encoding an MITF polypeptide to provide genetically modified iPSCs, wherein one or more of the nucleotide sequence encoding an OTX2 polypeptide, the nucleotide sequence encoding a PAX6 polypeptide, and the nucleotide sequence encoding an MITF polypeptide are operably linked to the promoter.
  • an inducible promoter e.g., a Tet-in
  • the genetically modified iPSCs are cultured under conditions sufficient for expression of the OTX2 polypeptide, the PAX6 polypeptide, and the MITF polypeptide in the genetically modified iPSCs, and differentiation of the genetically modified iPSCs into retinal pigment epithelial cells. In this manner RPE is generated.
  • the present disclosure provides a method of generating RPE from iPSCs, wherein the method includes introducing into the iPSCs a recombinant vector including an exogenous nucleic acid including a nucleotide sequence encoding an OTX2 polypeptide, an exogenous nucleic acid including a nucleotide sequence encoding a PAX6 polypeptide, and an exogenous nucleic acid including a nucleotide sequence encoding an MITF polypeptide to provide genetically modified iPSCs, wherein the recombinant vector is targeted to a genomic locus such as the AAVS1 locus or a CLYBL locus of the iPSCs.
  • the genetically modified iPSCs are cultured under conditions sufficient for expression of the OTX2 polypeptide, the PAX6 polypeptide, and the MITF polypeptide in the genetically modified iPSCs, and differentiation of the genetically modified iPSCs into retinal pigment epithelial cells. In this manner RPE is generated.
  • the present disclosure provides a method of generating RPE from iPSCs, wherein the method includes introducing into the iPSCs a recombinant vector including a promoter, an exogenous nucleic acid including a nucleotide sequence encoding an OTX2 polypeptide, an exogenous nucleic acid including a nucleotide sequence encoding a PAX6 polypeptide, and an exogenous nucleic acid including a nucleotide sequence encoding an MITF polypeptide to provide genetically modified iPSCs, wherein each of the nucleotide sequence encoding an OTX2 polypeptide, the nucleotide sequence encoding a PAX6 polypeptide, and the nucleotide sequence encoding an MITF polypeptide is operably linked to the promoter.
  • the genetically modified iPSCs are cultured under conditions sufficient for expression of the OTX2 polypeptide, the PAX6 polypeptide, and the MITF polypeptide in the genetically modified iPSCs, and differentiation of the genetically modified iPSCs into retinal pigment epithelial cells. In this manner RPE is generated.
  • the present disclosure provides RPE generated by the method of any one of aspects one through five as well as compositions including such RPE. Also provided, are methods of using the RPE and compositions prepared according to the methods of the present disclosure, e.g., screening methods including contacting the RPE with a test agent and assaying to determine the effect of the test agent on the RPE.
  • the present disclosure provides recombinant vectors useful in the methods disclosed herein, e.g., a recombinant vector including a promoter, a nucleic acid including a nucleotide sequence encoding an OTX2 polypeptide operably linked to the promoter, a nucleic acid including a nucleotide sequence encoding a PAX6 polypeptide operably linked to the promoter, and a nucleic acid including a nucleotide sequence encoding an MITF polypeptide operably linked to the promoter.
  • a recombinant vector including a promoter, a nucleic acid including a nucleotide sequence encoding an OTX2 polypeptide operably linked to the promoter, a nucleic acid including a nucleotide sequence encoding a PAX6 polypeptide operably linked to the promoter, and a nucleic acid including a nucleotide sequence encoding an MITF polypeptide operably linked
  • Figure 1 provides a vector map of a recombinant vector according to an aspect of the present disclosure.
  • Figure 2 provides images showing the stage of RPE development at 5 weeks
  • Figure 3 provides a schematic illustrating a method for RPE generation according to an aspect of the present disclosure.
  • Figure 4 provides a schematic illustrating a timeline for RPE generation according to an aspect of the present disclosure as well as images showing the stage of RPE development at different time points.
  • Figure 11 provides a cDNA and corresponding amino acid sequence for a human
  • OTX1 protein according to an aspect of the present disclosure (SEQ ID NOs.: 10-11).
  • Figure 12 provides a cDNA and corresponding amino acid sequence for a human
  • OTX2 protein according to an aspect of the present disclosure (SEQ ID NOs.: 12-13).
  • Figure 13 provides a cDNA and corresponding amino acid sequence for a human
  • PAX2 protein according to an aspect of the present disclosure (SEQ ID NOs.: 14-15).
  • Figure 14 provides a cDNA and corresponding amino acid sequence for a human
  • PAX6 protein according to an aspect of the present disclosure (SEQ ID NOs.: 16- 17).
  • Figure 15 provides a cDNA and corresponding amino acid sequence for a human
  • Figure 16 provides a summary of an RPE differentiation protocol adapted from Buchholz et al. Stem Cells Transl Med. 2013 May;2(5):384-93.
  • Figure 17 provides a CLYBL TALEN L vector map and sequence of a recombinant vector according to an aspect of the present disclosure.
  • Figure 18 provides a CLYBL TALEN R vector map and sequence of a recombinant vector according to an aspect of the present disclosure.
  • Figure 19 provides a CLYBL hOPM vector map and sequence of a recombinant vector according to an aspect of the present disclosure.
  • Figure 20 provides a AAVS1 TALEN L vector map and sequence of a recombinant vector according to an aspect of the present disclosure.
  • Figure 21 provides a AAVS1 TALEN R vector map and sequence of a recombinant vector according to an aspect of the present disclosure.
  • Figure 22 provides a AAVS hOPM vector map and sequence of a recombinant vector according to an aspect of the present disclosure.
  • Figure 23 provides a schematic of the hOPM construct.
  • Figure 24 provides images comparing results for RPE generation using the Leach et al. protocol and Zhu et al. protocol (left and middle, repsectively), and the methods described herein (right).
  • Figure 25 provides a schematic illustrating a timeline for RPE generation according to an aspect of the present disclosure as well as images showing the stage of RPE development at different time points.
  • Figure 26 provides schematics showing experimental set-up and results of
  • Figure 27 provides a schematic illustrating variations in optimized conditions
  • Figure 28 provides a schematic illustrating a timeline for variations in optimized conditions between cell lines according to an aspect of the present disclosure as well as images showing the stage of RPE development at different time points.
  • Figure 30 provides images showing the stage of RPE development at 60+ days for
  • Figure 32 provides images showing the stage of RPE development that are stained with generic tight junction proteins occluding and phalloidan.
  • Figure 33 provides images showing the stage of RPE development at day 60 for
  • Figure 35 provides images showing the stage of RPE development at day 60 for WTB and WTC.
  • Figure 36 provides gene expression results using ddPCR according to an aspect of the present disclosure.
  • pigmented layer of hexagonal cells present in vivo in mammals, just outside of the neurosensory retinal that is attached to the underlying choroid. These cells are densely packed with pigment granules, and shield the retinal from incoming light.
  • the retinal pigment epithelium also serves as the limiting transport factor that maintains the retinal environment by supplying small molecules such as amino acid, ascorbic acid and D-glucose while remaining a tight barrier to choroidal blood home substances.
  • the terms“Retinal Pigment Epithelium” and“RPE” are used herein to refer to a population of cells differentiated from iPSCs using the methods disclosed herein, wherein such cells exhibit one or more of pigmentation, epithelial morphology, and apical-basal polarity characteristic of naturally occurring RPE.
  • A“retinal pigment epithelial (RPE) cell e.g.
  • a cell differentiated from an iPSC using the methods disclosed herein can be recognized based on pigmentation, epithelial morphology, and apical-basal polarity.
  • RPE cells express, both at the mRNA and protein level, one or more of the following: MERTK, PAX6, MITF, RPE65, CRALBP, PEDF, OCCLUDIN, EZRIN, CLAUDIN-10, Bestrophin (e g., BEST1) and/or OTX2.
  • the RPE cells express, both at the mRNA and protein level, one or more of PAX6, MITF, and tyrosinase.
  • RPE cells do not express (at any detectable level) the embryonic stem cell markers Oct-4, nanog, or Rex-l. Specifically, expression of these genes is approximately 100-1000 fold lower in RPE cells than in ES cells or iPSC cells, when assessed by quantitative RT-PCR. Differentiated RPE cells also can be visually recognized by their cobblestone morphology and the initial appearance of pigment. In addition, differentiated RPE cells have trans-epithelial resistance/TER, and trans-epithelial potential/TEP across the monolayer (TERM 00 ohms cm 2 ; TEP>2 mV), transport fluid and CCh from apical to basal side, and regulate a polarized secretion of cytokines.
  • TERM 00 ohms cm 2 TEP>2 mV
  • embryonic stem cells refers to embryonic cells derived from the inner cell mass of blastocysts or morulae, optionally that have been serially passaged as cell lines.
  • the term includes cells isolated from one or more blastomeres of an embryo, preferably without destroying the remainder of the embryo.
  • the term also includes cells produced by somatic cell nuclear transfer.
  • “Human embryonic stem cells” (hES cells) includes embryonic cells derived from the inner cell mass of human blastocysts or morulae, optionally that have been serially passaged as cell lines.
  • the hES cells may be derived from fertilization of an egg cell with sperm or DNA, nuclear transfer, parthenogenesis, or by means to generate hES cells with homozygosity in the HLA region.
  • Human ES cells can be produced or derived from a zygote, blastomeres, or blastocyst-staged mammalian embryo produced by the fusion of a sperm and egg cell, nuclear transfer, parthenogenesis, or the reprogramming of chromatin and subsequent incorporation of the reprogrammed chromatin into a plasma membrane to produce an embryonic cell.
  • Human embryonic stem cells include, but are not limited to, MA01, MA09, ACT-4, No.
  • Human embryonic stem cells regardless of their source or the particular method used to produce them, can be identified based on (i) the ability to differentiate into cells of all three germ layers, (ii) expression of at least Oct-4 and alkaline phosphatase, and (iii) ability to produce teratomas when transplanted into immunocompromised animals.
  • pluripotent stem cells refers to stem cells that: (a) are capable of inducing teratomas when transplanted in immunodeficient (SCID) mice; (b) are capable of differentiating to cell types of all three germ layers (e.g., can differentiate to ectodermal, mesodermal, and endodermal cell types); and (c) express one or more markers of embryonic stem cells (e.g., express Oct 4, alkaline phosphatase, SSEA-3 surface antigen, SSEA-4 surface antigen, nanog, TRA-l-60, TRA-1-81, SOX2, REX1, etc.), but that cannot form an embryo and the
  • extraembryonic membranes are not totipotent.
  • Exemplary pluripotent stem cells include embryonic stem cells derived from the inner cell mass (ICM) of blastocyst stage embryos, as well as embryonic stem cells derived from one or more blastomeres of a cleavage stage or morula stage embryo (optionally without destroying the remainder of the embryo). These embryonic stem cells can be generated from embryonic material produced by fertilization or by asexual means, including somatic cell nuclear transfer (SCNT), parthenogenesis, and androgenesis. PSCs alone cannot develop into a fetal or adult animal when transplanted in utero because they lack the potential to contribute to all
  • extraembryonic tissue e.g., placenta in vivo or trophoblast in vitro.
  • Pluripotent stem cells also include“induced pluripotent stem cells (iPSCs)”
  • iPSCs can be generated using fetal, postnatal, newborn, juvenile, or adult somatic cells.
  • factors that can be used to reprogram somatic cells to pluripotent stem cells include, for example, Oct4 (sometimes referred to as Oct 3/4), Sox2, c-Myc, and Klf4, Nanog, and Lin28.
  • somatic cells are reprogrammed by expressing at least two reprogramming factors, at least three reprogramming factors, or four reprogramming factors to reprogram a somatic cell to a pluripotent stem cell.
  • iPSCs are similar in properties to embryonic stem cells.
  • reprogramming factors refers to one or more biologically active factors that act on a cell to alter transcription, thereby reprogramming a cell to multipotency or to pluripotency.
  • Reprogramming factors may be provided to cells, e.g. cells from an individual with a family history or genetic make-up of interest for an RPE related disease, e.g., age-related macular degeneration; individually or as a single composition, that is, as a premixed composition, of reprogramming factors.
  • the factors may be provided at the same molar ratio or at different molar ratios.
  • the factors may be provided once or multiple times in the course of culturing the cells.
  • the reprogramming factor is a transcription factor, including without limitation, Oct3/4; Sox2; Klf4; c-Myc; Nanog; and Lin-28.
  • Somatic cells are contacted with reprogramming factors, as defined above, in a
  • Reprogramming factors may be provided to the somatic cells individually or as a single composition, that is, as a premixed composition, of reprogramming factors.
  • the reprogramming factors are provided as a plurality of coding sequences on a vector.
  • Genes may be introduced into the somatic cells or the iPSCs derived therefrom for a variety of purposes, e.g. to replace genes having a loss of function mutation, provide marker genes, etc.
  • vectors are introduced that express antisense mRNA or ribozymes, thereby blocking expression of an undesired gene.
  • Other methods of gene therapy are the introduction of drug resistance genes to enable normal progenitor cells to have an advantage and be subject to selective pressure, for example the multiple drug resistance gene (MDR), or anti-apoptosis genes, such as bcl-2.
  • MDR multiple drug resistance gene
  • anti-apoptosis genes such as bcl-2.
  • Various techniques known in the art may be used to introduce nucleic acids into the target cells, e.g. electroporation, calcium precipitated DNA, fusion, transfection, lipofection, infection and the like, as discussed above. The particular manner in which the DNA is introduced is not critical to the practice of the invention.
  • the term“differentiation” refers to the process whereby relatively unspecialized cells (such as embryonic stem cells or other stem cells) acquire specialized structural and/or functional features characteristic of mature cells.
  • differentiate refers to this process. Typically, during differentiation, cellular structure alters and tissue-specific proteins appear.
  • the term“expand” refers to a process by which the number or
  • the terms “expansion” or“expanded” refers to this process.
  • the terms“proliferate,” “proliferation” or“proliferated” may be used interchangeably with the words “expand,”“expansion”, or“expanded.” Typically, during an expansion phase, the cells do not differentiate to form mature cells, but divide to form more cells.
  • growth factor refers to a substance that promotes cell growth, survival, and/or differentiation.
  • Growth factors include molecules that function as growth stimulators (mitogens), factors that stimulate cell migration, factors that function as chemotactic agents or inhibit cell migration or invasion of tumor cells, factors that modulate differentiated functions of cells, factors involved in apoptosis, or factors that promote survival of cells without influencing growth and differentiation.
  • growth factors are a fibroblast growth factor (such as FGF-2), epidermal growth factor (EGF), cilliary neurotrophic factor (CNTF), and nerve growth factor (NGF), and activin-A.
  • “growth medium” and“expansion medium” refer to a
  • the cells are stem cells, such as iPSCs.
  • Growth media generally include a carbon source, a nitrogen source and a buffer to maintain pH.
  • growth medium contains a minimal essential media, such as DMEM, supplemented with various nutrients to enhance stem cell growth. Additionally, the minimal essential media may be supplemented with additives such as horse, calf or fetal bovine serum.
  • A“host cell,” as used herein, denotes an in vivo or in vitro cell (e.g., a eukaryotic cell cultured as a unicellular entity), which eukaryotic cell can be, or has been, used as recipients for a nucleic acid (e.g., an exogenous nucleic acid), and include the progeny of the original cell which has been genetically modified by the nucleic acid. It is understood that the progeny of a single cell may not necessarily be completely identical in morphology or in genomic or total DNA complement as the original parent, due to natural, accidental, or deliberate mutation.
  • the term“isolated” with reference to a cell refers to a cell that is in an environment different from that in which the cell naturally occurs, e.g., where the cell naturally occurs in a multicellular organism, and the cell is removed from the multicellular organism, the cell is“isolated.”
  • An isolated genetically modified host cell can be present in a mixed population of genetically modified host cells, or in a mixed population comprising genetically modified host cells and host cells that are not genetically modified.
  • an isolated genetically modified host cell can be present in a mixed population of genetically modified host cells in vitro, or in a mixed in vitro population comprising genetically modified host cells and host cells that are not genetically modified.
  • transcription factor refers to a protein that targets a
  • the term“genetic modification,” as used herein, refers to a permanent or transient genetic change induced in a cell following introduction of a nucleic acid (i.e., exogenous nucleic acid) into the cell.
  • Genetic change can be accomplished by incorporation of exogenous nucleic acid into the genome of the host cell, or by transient or stable maintenance of the exogenous nucleic acid as an extrachromosomal element.
  • a permanent genetic change can be achieved by introduction of exogenous nucleic acid into the genome of the cell. Suitable methods of genetic modification include viral infection, transfection, conjugation, protoplast fusion, electroporation, particle gun technology, calcium phosphate precipitation, direct microinjection, and the like.
  • exogenous nucleic acid refers to a nucleic acid that is not normally or naturally found in and/or produced by a cell in nature, and/or that is introduced into the cell (e.g., by electroporation, transfection, infection, lipofection, or any other means of introducing a nucleic acid into a cell).
  • polynucleotide and“nucleic acid,” used interchangeably herein, refer to a polymeric form of nucleotides of any length, either ribonucleotides or
  • deoxy ribonucleotides include, but is not limited to, single-, double-, or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or a polymer comprising purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural, or derivatized nucleotide bases.
  • Oligonucleotide generally refers to polynucleotides of between about 5 and about 100 nucleotides of single- or double-stranded DNA. However, for the purposes of this disclosure, there is no upper limit to the length of an oligonucleotide.
  • Oligonucleotides are also known as oligomers or oligos and may be isolated from genes, or chemically synthesized by methods known in the art. It will be understood that when a nucleotide sequence is represented by a DNA sequence (i.e., A, T, G, C), this also includes an RNA sequence (i.e., A, U, G, C) in which“U” replaces“T.” [0071] A first nucleic acid sequence is“operably linked” with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For instance, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence.
  • polypeptide refers to a polymeric form of amino acids of any length, which can include coded and non- coded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides having modified peptide backbones.
  • the term includes fusion proteins, including, but not limited to, fusion proteins with a heterologous amino acid sequence, fusions with heterologous and homologous leader sequences, with or without N-terminal methionine residues; immunologically tagged proteins; and the like.
  • a polynucleotide or polypeptide has a certain percent“sequence identity” to another polynucleotide or polypeptide, meaning that, when aligned, that percentage of bases or amino acids are the same, and in the same relative position, when comparing the two sequences. Sequence similarity can be determined in a number of different manners. To determine sequence identity, sequences can be aligned using the methods and computer programs, including BLAST, available over the world wide web at ncbi.nlm.nih.gov/BLAST. See, e.g., Altschul et al. (1990), J. Mol. Biol.
  • Polypeptide described herein may include one or more conservative amino acid
  • amino acids that are considered to be conservative substitutions for one another are well known to one of ordinary skill in the art.
  • a non-conservative amino acid substitution can result from changes in: (a) the
  • substitutions generally expected to produce the greatest changes in protein properties are those in which: (a) a hydrophilic residue is substituted for (or by) a hydrophobic residue; (b) a proline is substituted for (or by) any other residue; (c) a residue having a bulky side chain, e.g., phenylalanine, is substituted for (or by) one not having a side chain, e.g., glycine; or (d) a residue having an electropositive side chain, e.g., lysyl, arginyl, or histadyl, is substituted for (or by) an electronegative residue, e.g., glutamyl or aspartyl.
  • a hydrophilic residue is substituted for (or by) a hydrophobic residue
  • a proline is substituted for (or by) any other residue
  • a residue having a bulky side chain e.g., phenylalanine
  • an electropositive side chain e
  • Variant amino acid sequences may, for example, be 80, 90 or even 95 or 98%
  • cDNA refers to a DNA that is complementary or identical to an mRNA, in either single stranded or double stranded form.
  • the terms“encode” and“encoding” refer to the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (for example, rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom.
  • a gene encodes a protein if transcription and translation of mRNA produced by that gene produces the protein in a cell or other biological system.
  • coding strand the nucleotide sequence of which is identical to the mRNA sequence and is usually provided in sequence listings
  • non-coding strand used as the template for transcription, of a gene or cDNA
  • a“nucleotide sequence encoding an amino acid sequence” includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. Nucleotide sequences that encode proteins and RNA may include introns.
  • the term“recombinant”, refers to a nucleic acid or polypeptide
  • molecule that has a sequence that is not naturally occurring or has a sequence that is made by an artificial combination of two otherwise separated segments of sequence.
  • This artificial combination can be accomplished by chemical synthesis of polypeptide or nucleic acid molecules, or by the artificial manipulation of isolated segments of nucleic acid molecules, such as by genetic engineering techniques.
  • the terms“treatment,”“treating,” and the like refer to obtaining a desired pharmacologic and/or physiologic effect.
  • the effect may be prophylactic in terms of completely or partially preventing a disease or symptom thereof and/or may be therapeutic in terms of a partial or complete cure for a disease and/or adverse effect attributable to the disease.
  • “Treatment,” as used herein covers any treatment of a disease in a mammal, particularly in a human, and includes: (a) preventing the disease from occurring in a subject which may be predisposed to the disease but has not yet been diagnosed as having it; (b) inhibiting the disease, i.e., arresting its development; and (c) relieving the disease, i.e., causing regression of the disease.
  • the terms“individual,”“subject,”“host,” and“patient,” used interchangeably herein, refer to a mammal, including, but not limited to, murines (rats, mice), non-human primates, humans, canines, felines, ungulates (e.g., equines, bovines, ovines, porcines, caprines), etc.
  • A“therapeutically effective amount” or“efficacious amount” refers to the amount of a compound or a number of cells that, when administered to a mammal or other subject for treating a disease, is sufficient to effect such treatment for the disease.
  • the “therapeutically effective amount” will vary depending on the compound or the cell, the disease and its severity and the age, weight, etc., of the subject to be treated.
  • “AAVS1” is an abbreviation for a site in the human genome that was defined as a preferred integration site of the adeno-associated virus. This“safe harbor” site is in the middle of PPP1R12C gene (see, e.g., DeKelver et al, Genome Research 2010).
  • gene editing tool is used herein to refer to one or more components of a gene editing system.
  • RPE retinal pigment epithelium
  • iPSCs induced pluripotent stem cells
  • the methods generally involve: introducing into the iPSCs an exogenous nucleic acid including a nucleotide sequence encoding an orthodenticle homeobox 2 (OTX2) polypeptide, an exogenous nucleic acid including a nucleotide sequence encoding a paired box protein (PAX6) polypeptide, and an exogenous nucleic acid including a nucleotide sequence encoding a microphthalmia-associated transcription factor (MITF) polypeptide to provide genetically modified iPSCs.
  • OTX2 orthodenticle homeobox 2
  • PAX6 paired box protein
  • MITF microphthalmia-associated transcription factor
  • the genetically modified iPSCs are cultured under conditions sufficient for expression of the OTX2 polypeptide, the PAX6 polypeptide, and the MITF polypeptide in the genetically modified iPSCs, and under conditions sufficient for differentiation of the genetically modified iPSCs into retinal pigment epithelial cells.
  • the methods specifically exclude the introduction into the iPSCs or genetically modified iPSCs of a transcription factor other than an OTX2 polypeptide, a PAX6 polypeptide, and/or an MITF polypeptide, or an exogenous nucleic acid comprising a nucleotide sequence encoding a transcription factor other than an OTX2 polypeptide, a PAX6 polypeptide, and/or an MITF polypeptide.
  • RPE having desired characteristics as described herein can be generated via the introduction of a limited number of specific transcription factors into iPSCs, specifically via the introduction of OTX2, PAX6 and MITF and without the introduction of transcription factors other than OTX2, PAX6 and MITF.
  • the methods specifically exclude introducing into the iPSCs or genetically modified iPSCs an exogenous nucleic acid comprising a tyrosinase enhancer.
  • suitable iPSCs e.g., genetically modified iPSCs as described herein, are cultured in a suitable growth medium prior to exposing the cells to differentiation conditions on Day 0 of the differentiation protocol.
  • a suitable grown medium may be, for example, a feeder free maintenance medium for human ES and iPSCs.
  • Exemplary media includes mTeSRTM media available from Stemcell Technologies, e.g., Catalog #85850,
  • the growth medium may be supplemented with a selective Rho-associated kinase (ROCK) inhibitor, e.g., Y-27632. See, e.g., Lai et al. Cell Reprogram. 2010 Dec; 12(6): 641-653.
  • iPSCs may be seeded at a suitable time prior to introduction of a suitable differentiation medium, e.g., at Day -2 of the differentiation protocol, at a density allowing the culture to grow to confluence at Day 0.
  • a suitable differentiation medium e.g., at Day -2 of the differentiation protocol, at a density allowing the culture to grow to confluence at Day 0.
  • iPSCs may be plated at a density of from about 35,000 cells/cm 2 growth area to about 55,000 cells/cm 2 of growth area, e.g., from about 40,000 cells/cm 2 growth area to about 50,000 cells/cm 2 growth area, such as about 44,000 cells/cm 2 growth area.
  • the plating density e.g., at Day -2, may be adjusted such that the culture grows to confluence at Day 0.
  • a suitable differentiation medium e.g., a neural induction medium
  • a suitable neural induction medium is then added to the culture (e.g., on Day 0 of the differentiation protocol).
  • Suitable neural induction media are described in the examples section of the present disclosure as well as in, e.g., Yan et al. Stem Cells Transl Med. 2013 Nov; 2(11): 862-870; and Chambers, S.M. et al. (2009) Nat. Biotechnol. 27:275-280.
  • the cells may be cultured in a suitable neural induction medium for a period of time prior to culturing the genetically modified iPSCs under conditions sufficient for expression of an OTX2 polypeptide, a PAX6 polypeptide, and an MITF polypeptide from exogenous nucleic acids in the genetically modified iPSCs.
  • This period of time may range from about 1 to about 7 days, e.g., from about 2 to about 6 days, or about 3 to about 5 days, such as about 4 days.
  • expression of the OTX2 polypeptide, the PAX6 polypeptide, and the MITF polypeptide from exogenous nucleic acids in the genetically modified iPSCs is induced at Day 4 of culture in a suitable neural induction medium.
  • expression of the OTX2 polypeptide, the PAX6 polypeptide, and the MITF polypeptide from exogenous nucleic acids in the genetically modified iPSCs is under the control of a Tet-inducible promoter
  • expression may be induced via the addition of tetracycline or a derivative or analogue thereof, e.g., doxycycline, to the neural induction medium.
  • the cells may be cultured (e.g., starting on about Day 5 to about Day 7, e.g., Day 6) in a suitable maintenance medium, e.g., in the presence of tetracycline or a derivative or analogue thereof, e.g., doxycycline, to maintain expression of the OTX2 polypeptide, the PAX6 polypeptide, and the MITF polypeptide from exogenous nucleic acids in the differentiating cells.
  • a suitable maintenance medium e.g., in the presence of tetracycline or a derivative or analogue thereof, e.g., doxycycline
  • RPE maintenance medium differentiating RPE cells are described in the examples section (RPE maintenance medium) of the present disclosure as well as in, e.g., Fronk et al. J Tissue Eng. 2016 Jan-Dec; 7:2041731416650838.
  • the culture medium may be changed to a suitable chemically defined, serum-free hematopoietic cell medium, e.g., X-vivoTM 10 available from Lonza Group AG under catalog numbers: 04-380Q, 04-743Q, BE04-743Q, and BE02-055Q, e.g., in the presence of tetracycline or a derivative or analogue thereof, e.g., doxycycline, to maintain expression of the OTX2 polypeptide, the PAX6 polypeptide, and the MITF polypeptide from exogenous nucleic acids in the differentiating cells.
  • a suitable chemically defined, serum-free hematopoietic cell medium e.g., X-vivoTM 10 available from Lonza Group AG under catalog numbers: 04-380Q, 04-743Q, BE04-743Q, and BE02-055Q, e.g., in the presence of tetracycline or a derivative or analogue thereof, e.g
  • the medium may be changed to a suitable chemically defined, serum-free hematopoietic cell medium, e.g., X-vivoTM 10 as described above, without the addition of tetracycline or a derivative or analogue thereof.
  • a suitable chemically defined, serum-free hematopoietic cell medium e.g., X-vivoTM 10 as described above, without the addition of tetracycline or a derivative or analogue thereof.
  • iPSCs may be cultured in a suitable medium
  • differentiation medium e.g., a neural induction medium
  • a neural induction medium for a period of time prior to culturing the genetically modified iPSCs under conditions sufficient for expression of an OTX2 polypeptide, a PAX6 polypeptide, and an MITF polypeptide from exogenous nucleic acids in the genetically modified iPSCs.
  • Delaying expression of the OTX2 polypeptide, the PAX6 polypeptide, and the MITF polypeptide e.g., by delaying induction of a Tet-inducible promoter operably linked to exogenous nucleic acids including nucleotide sequences encoding the OTX2 polypeptide, the PAX6 polypeptide, and the MITF polypeptide, for a period of time while the cells are exposed to a suitable differentiation medium has been found to provide for improved generation of RPE.
  • a step of culturing genetically modified iPSCs under conditions sufficient for expression of the OTX2 polypeptide, the PAX6 polypeptide, and the MITF polypeptide in the genetically modified iPSCs includes a step of culturing the genetically modified iPSCs under differentiation conditions, e.g., in a suitable differentiation medium, e.g., a neural induction medium, prior to culturing the genetically modified iPSCs under conditions sufficient for expression of the OTX2 polypeptide, the PAX6 polypeptide, and the MITF polypeptide in the genetically modified iPSCs, e.g., prior to induction of a Tet-inducible promoter operably linked to exogenous nucleic acids including nucleotide sequences encoding the OTX2 polypeptide, the PAX6 polypeptide, and the MITF polypeptide.
  • This prior culturing step may be for a suitable period of time, e.g
  • exogenous nucleic acid including a nucleotide sequence encoding an OTX2 polypeptide, an exogenous nucleic acid including a nucleotide sequence encoding a PAX6 polypeptide, and an exogenous nucleic acid including a nucleotide sequence encoding an MITF polypeptide is sufficient to differentiate iPSCs into RPE cells and for the generation of RPE.
  • an iPSC can be differentiated to become an RPE cell without the need for introducing any other transcription factor (or nucleic acid encoding same) into the iPSC.
  • an iPSC can be differentiated to become an RPE cell without the need for introducing nucleic acids encoding other factors that may influence stem cell to RPE differentiation, or RPE function, or physiology, or pathology, into the iPSC.
  • a subject method does not require and does not involve introducing into a iPSC a nucleic acid that encodes any one or more of the following: TFEC (NM_0l2252, NP_036384.
  • LHX2 (NM_004789, NP_004780.3 ), VMD2 (NM 001139443.1, NP_00l 132915.1), CFTR (NM_000492.3, NP_000483.3 ), RPE65 (NM_000329.2, NP_000320. l), MFRP (NM_03l433.3, NP_l l362l. l ), CTRP5 (AF329841.1, AAK17965.1), CFH
  • NM 001302688.1, ⁇ _001289617.1 APOB
  • APOB NM_000384.2, NP_000375.2
  • mTOR NM_004958.3, NP_004949.l
  • FOXOl NM_0020l5.3, NP_002006.2
  • AMPK NM_00625l.5, NP_006242.5
  • SIRT1-6 NM_0l2238.4, NP_036370.2; AY030277.1, AAK51133.1; NM_0l2239.5, NP_03637l. l; AF083109.1,
  • NP_00l 020537.2) CFI (NM_000204.4 , NP_000l95.2)
  • TLR3 NM_003265.2, NP_003256.1
  • TLR4 NM_003266.3, NP_003257. l
  • APP NM_000484.3, NP_000475.
  • CD46 NM_002389.4, NP_002380.3
  • BACE1 NM_001207048.1, NP_00l 193977.1
  • ELOVL4 NM_022726.3, NP_073563. l
  • recombinant vectors according to the present disclosure may exclude a nucleic acid that encodes any one or more of the following: TFEC, LHX2, VMD2, CFTR, RPE65, MFRP, CTRP5, CFH, C3, C2B, APOE, APOB, mTOR, FOXO, AMPK, SIRT1-6, HTRP1, ABCA4, TIMP3, VEGFA, CFI, TLR3, TLR4, APP, CD46, BACE1, ELOVL4, ADAM 10, CD55, CD59, and ARMS2.
  • a subject method does not involve forced expression of a polypeptide comprising an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 95%, 97%, or at least 99% amino acid sequence identity, or any other percent identity from at least 70% to 100%, to a TFEC, LHX2, VMD2, CFTR, RPE65, MFRP, CTRP5, CFH, C3, C2B, APOE, APOB, mTOR, FOXO, AMPK, SIRT1-6, HTRP1, ABCA4, TIMP3, VEGFA, CFI, TLR3, TLR4, APP, CD46, BACE1, ELOVL4, ADAM 10, CD55, CD59, or ARMS2 polypeptide.
  • a subject method can involve forced expression of an OTX2 polypeptide, a PAX6 polypeptide, and an MITF polypeptide in an iPSC and/or a differentiating RPE cell. Forced expression of a polypeptide can include introducing an expression vector encoding polypeptides of interest into cells. In some embodiments, a subject method does not involve forced expression of any polypeptide other than an OTX2 polypeptide, a PAX6 polypeptide, and an MITF polypeptide in the iPSC and/or a differentiating RPE cell.
  • a subject method does not involved forced expression of any of a TFEC, LHX2, VMD2, CFTR, RPE65, MFRP, CTRP5, CFH, C3, C2B, APOE, APOB, mTOR, FOXO, AMPK, SIRT1-6, HTRP1, ABCA4, TIMP3, VEGFA, CFI, TLR3, TLR4, APP, CD46, BACE1, ELOVL4, ADAM 10, CD55, CD59, or ARMS 2 polypeptide.
  • RPE cell differentiated from an iPSC using the methods disclosed herein can be recognized based on pigmentation, epithelial morphology, and apical-basal polarity.
  • RPE cells express, both at the mRNA and protein level, one or more of the following: Pax6, MITF, RPE65 (NM_000329.2, NP_000320.l), CRALBP
  • the RPE cells express, both at the mRNA and protein level, one or more of Pax-6, MitF, and tyrosinase (NM_000372.4, NP_000363. l). RPE cells do not express (at any detectable level) the embryonic stem cell markers Oct-4, nanog, or Rex-l.
  • expression of these genes is approximately 100- 1000 fold lower in RPE cells than in ES cells or iPSC cells, when assessed by quantitative RT-PCR.
  • Differentiated RPE cells also can be visually recognized by their cobblestone morphology and the initial appearance of pigment.
  • differentiated RPE cells have trans-epithelial resistance/TER, and trans-epithelial potential/TEP across the monolayer (TERM 00 ohms cm 2 ; TEP>2 mV), transport fluid and CCh from apical to basal side, and regulate a polarized secretion of cytokines.
  • cell-specific markers and differentiation-specific markers can be detected using any suitable immunological technique, such as fluorescence- activated cell sorting (FACS) for cell-surface markers, immunohistochemistry (for example, of fixed cells or tissue sections) for intracellular or cell-surface markers, Western blot analysis of cellular extracts, and enzyme-linked immunoassay, for cellular extracts or products secreted into the medium.
  • FACS fluorescence- activated cell sorting
  • immunohistochemistry for example, of fixed cells or tissue sections
  • Western blot analysis of cellular extracts for example, of fixed cells or tissue sections
  • enzyme-linked immunoassay for cellular extracts or products secreted into the medium.
  • an antigen a marker
  • Expression of an antigen (a marker) by a cell is said to be“antibody-detectable” if a significantly detectable amount of antibody will bind to the antigen in a standard immunocytochemistry or flow cytometry assay, optionally after fixation of the cells, and optionally using a labeled secondary antibody or other conjugate (such as a biotin-avidin conjugate) to amplify labeling.
  • a labeled secondary antibody or other conjugate such as a biotin-avidin conjugate
  • the expression of cell-specific and differentiation-specific gene products can also be detected at the mRNA level by Northern blot analysis, dot-blot hybridization analysis, or by a reverse transcription polymerase chain reaction (RT-PCR) using sequence-specific primers in standard amplification methods.
  • Expression at the mRNA level is said to be“detectable” according to a standard assay if the performance of the assay on cell samples according to standard procedures in a typical controlled experiment results in clearly discemable hybridization or amplification product.
  • cell-specific e.g., RPE cell-specific
  • differentiation-specific markers as detected at the protein or mRNA level is considered positive if the level is at least 2-fold, or more than 10- or 50-fold above that of a control cell, such as a pluripotent embryonic stem cell, a fibroblast, or other unrelated cell type.
  • OTX2 orthodenticle homeobox 2 polypeptides are known in the art, and any OTX2 polypeptide that retains OTX2 activity (or a nucleic acid encoding such polypeptide) is suitable for use.
  • An OTX2 polypeptide that retains OTX2 activity is also referred to as a“biologically active OTX2 polypeptide.”
  • a suitable OTX2 polypeptide that retains OTX2 activity is one that: (i) binds and activates the DOPAchrome tautomerase (DCT) gene (or homolog thereof) promoter in RPE cells; and/or (ii) binds and synergistically activates the BEST1 (or homolog thereof) promoter in combination with SOX9 (or homolog thereof) and MITF (or homolog thereof). See, e.g., Takeda et al. Biochem Biophys Res Commun. 2003 Jan.
  • OTX2 amino acid sequences can be found in, e.g., GenBank Accession Nos/NCBI Reference Sequences: AAD31385.1, ⁇ _001257452.1, ⁇ _001257453.1, ⁇ _001257454.1, ⁇ _068374.1, and
  • a suitable OTX2 polypeptide retains OTX2 activity and comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 95%, 97%, or at least 99% amino acid sequence identity, or any other percent identity from at least 70% to 100%, to the OTX2 amino acid sequence depicted in Figure 12 or to the amino acid sequence provided by one of AAD31385.1, ⁇ _001257452.1, NP_00l257453. l, NP_00l257454. l, NP_068374. l, and NP_758840. l.
  • the OTX2 gene is conserved in chimpanzee, dog, cow, mouse, rat, and
  • zebrafish See, e.g., XP_001163043.2, XP_003435187.1, NP_00l 180130.1, ⁇ _001273410.1, XP_006251897.1, and NP_571326.1.
  • OTX2 polypeptide-encoding nucleic acids can be found in, e.g., GenBank Accession Nos/NCBI Reference Sequences: NM_00l270523. l. NM_00l270524. l, NM_001270525.1,
  • PAX6 (paired box 6) polypeptides are known in the art, and any PAX6
  • a PAX6 polypeptide that retains PAX6 activity is suitable for use.
  • a PAX6 polypeptide that retains PAX6 activity is also referred to as a“biologically active PAX6 polypeptide.”
  • a suitable PAX6 polypeptide that retains PAX6 activity is one that: (i) includes two DNA binding domains (a paired domain (PD) at the N-terminus and a paired-like homeodomain (HD), a glycine-rich region that links the two DNA binding domains, and a transactivation domain at the C-terminus; and (ii) binds the PAX6-specific binding site Tsp2Asl
  • a suitable PAX6 polypeptide retains PAX6 activity and comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 95%, 97%, or at least 99% amino acid sequence identity, or any other percent identity from at least 70% to 100%, to the PAX6 amino acid sequence depicted in Figure 13 or to the amino acid sequence provided by one ofNP_00027l. l, NP_00l 121084.1, NP_00l24539l. l, NP_00l245392.1, NP_00l245393. l, NP_001245394.1, NP_00l297087.l,
  • PAX6 gene is conserved in chimpanzee, Rhesus monkey, cow, mouse, rat, chicken, zebrafish, fruit fly, mosquito, C.elegans, and frog. See, e.g.,
  • PAX6 polypeptide-encoding nucleic acids can be found in, e.g., GenBank Accession Nos/NCBI Reference Sequences:
  • NM_001258464.1 NM_001258465.1, ⁇ _001310158.1, ⁇ _001310159.1, ⁇ _001310160.1, ⁇ _001310161.1, and ⁇ _001604.5.
  • MITF Microphthalmia-associated transcription factor
  • MITF polypeptides are known in the art, and any MITF polypeptide that retains MITF activity (or a nucleic acid encoding such polypeptide) is suitable for use.
  • a MITF polypeptide that retains MITF activity is also referred to as a“biologically active MITF polypeptide.”
  • a suitable MITF polypeptide that retains MITF activity is one that: (i); is capable of forming both a homodimer and a heterodimer and (ii) binds consensus M-box (TCATGTG (SEQ ID NO:4)) and E-box (CACGTG (SEQ ID NO:5)) sequences. See, e.g., Pogenberg et al.
  • MITF amino acid sequences can be found in, e.g., GenBank Accession Nos/NCBI Reference Sequences: NP_000239.l, ⁇ _001171896.1, NP_00l 171897.1, ⁇ _001341533.1, ⁇ _001341534.1,
  • NP_93780l.l NP_937802. l
  • NP_937820. l NP_93782l.2.
  • a suitable MITF polypeptide retains MITF activity and comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 95%, 97%, or at least 99% amino acid sequence identity, or any other percent identity from at least 70% to 100%, to the MITF amino acid sequence depicted in Figure 14 or to the amino acid sequence provided by one ofNP_000239. l, NP_00l 171896.1 , NP_00l 171897.1, ⁇ _001341533.1,
  • NP_001341534.1 ⁇ _001341535.1
  • ⁇ _001341536.1 ⁇ _001341537.1
  • NP_006713.1 NP_93780l. l, NP_937802. l, NP_937820. l, and NP_93782l.2.
  • the MITF gene is conserved in chimpanzee, Rhesus monkey, dog, cow, mouse, rat, chicken, zebrafish, and frog. See, e.g., XP_00l 138876.1,
  • MITF polypeptide-encoding nucleic acids can be found in, e.g., NM_000248.3,
  • introduction of an exogenous nucleic acid including a nucleotide sequence encoding an OTX2 polypeptide, an exogenous nucleic acid including a nucleotide sequence encoding a PAX6 polypeptide, and an exogenous nucleic acid including a nucleotide sequence encoding an MITF polypeptide into an iPSC is achieved by genetic modification of the iPSC with an exogenous nucleic acid including a nucleotide sequence encoding an OTX2 polypeptide, an exogenous nucleic acid including a nucleotide sequence encoding a PAX6 polypeptide, and an exogenous nucleic acid including a nucleotide sequence encoding an MITF polypeptide.
  • exogenous nucleic acid comprising a nucleotide sequence encoding an OTX2 polypeptide is also referred to herein as“an exogenous OTX2 nucleic acid.”
  • An exogenous nucleic acid comprising a nucleotide sequence encoding a PAX6 polypeptide is also referred to herein as“an exogenous PAX6 nucleic acid.”
  • An exogenous nucleic acid comprising a nucleotide sequence encoding a MITF polypeptide is also referred to herein as“an exogenous MITF nucleic acid.”
  • exogenous OTX2 nucleic acid, exogenous PAX6 nucleic acid, and/or exogenous MITF nucleic acid can be a recombinant expression vector, where suitable vectors include, e.g., recombinant retroviruses, lentiviruses, and adenoviruses;
  • the exogenous OTX2 nucleic acid, exogenous PAX6 nucleic acid, and/or exogenous MITF nucleic acid is integrated into the genome of a host iPSC cell and its progeny.
  • the exogenous OTX2 nucleic acid, exogenous PAX6 nucleic acid, and/or exogenous MITF nucleic acid persists in an episomal state in the host iPSC cell and its progeny.
  • an endogenous, natural version of the OTX2-encoding nucleic acid, PAX6-encoding nucleic acid, and/or MITF-encoding nucleic acid may already exist in the cell but an additional“exogenous gene” is added to the host iPSC cell to increase OTX2, PAX6, and/or MITF polypeptide expression.
  • the exogenous OTX2 nucleic acid, exogenous PAX6 nucleic acid, and/or exogenous MITF nucleic acid encodes an OTX2 polypeptide, PAX6 polypeptide, and/or MITF polypeptide, respectively, having an amino acid sequence that differs by one or more amino acids from an OTX2 polypeptide, PAX6 polypeptide, and/or MITF polypeptide encoded by an endogenous OTX2-encoding nucleic acid, endogenous PAX6-encoding nucleic acid, and/or endogenous MITF-encoding nucleic acid, respectively, within the host iPSC cell.
  • an exogenous OTX2 nucleic acid, exogenous PAX6 nucleic acid, and/or exogenous MITF nucleic acid is introduced into a single iPSC (e.g., a single iPSC host cell) in vitro.
  • an exogenous OTX2 nucleic acid, exogenous PAX6 nucleic acid, and/or exogenous MITF nucleic acid is introduced into a population of iPSCs (e.g., a population of host iPSCs) in vitro.
  • the an exogenous OTX2 nucleic acid, exogenous PAX6 nucleic acid, and/or exogenous MITF nucleic acid can be introduced into greater than 20% of the total population of cells somatic cells, e.g., 25%, 30%, 35%, 40%, 44%, 50%, 57%, 62%, 70%, 74%, 75%, 80%, 90%, or other percent of cells greater than 20%.
  • suitable mammalian expression vectors include, but are not limited to: recombinant viruses, nucleic acid vectors, such as plasmids, bacterial artificial chromosomes, yeast artificial chromosomes, human artificial chromosomes, cDNA, cRNA, and polymerase chain reaction (PCR) product expression cassettes.
  • nucleic acid vectors such as plasmids, bacterial artificial chromosomes, yeast artificial chromosomes, human artificial chromosomes, cDNA, cRNA, and polymerase chain reaction (PCR) product expression cassettes.
  • Suitable promoters for driving expression of an OTX2-encoding nucleotide sequence, PAX6-encoding nucleotide sequence, and/or MITF-encoding nucleotide sequence include, but are not limited to, retroviral long terminal repeat (LTR) elements; constitutive promoters such as CMV, HSV1-TK, SV40, EF-la, ⁇ - actin; phosphoglycerol kinase (PGK), and inducible promoters, such as those containing Tet-operator elements.
  • LTR retroviral long terminal repeat
  • constitutive promoters such as CMV, HSV1-TK, SV40, EF-la, ⁇ - actin
  • PGK phosphoglycerol kinase
  • inducible promoters such as those containing Tet-operator elements.
  • the mammalian expression vector encodes, in addition to an exogenous OTX2 polypeptide, PAX6 polypeptide and/or MITF polypeptide, a marker gene that facilitates identification or selection of cells that have been transfected or infected.
  • marker genes include, but are not limited to, genes encoding fluorescent proteins, e.g., enhanced green fluorescent protein, DS-Red, yellow fluorescent protein, and cyanofluorescent protein; and genes encoding proteins conferring resistance to a selection agent, e.g., the neomycin resistance gene, and the blasticidin resistance gene.
  • recombinant viruses include, but are not limited, to retroviruses (including lentiviruses); adenoviruses; and adeno-associated viruses.
  • retroviruses including lentiviruses
  • adenoviruses including lentiviruses
  • adeno-associated viruses include murine moloney leukemia virus (MMLV);
  • retroviruses may also be used, e.g., Avian Leukosis Virus, Bovine Leukemia Virus, Murine Leukemia Virus (MLV), Mink-Cell focus- inducing Virus, Murine Sarcoma Virus, Reticuloendotheliosis virus, Gibbon Abe Leukemia Virus, Mason Pfizer Monkey Virus, or Rous Sarcoma Virus, see, e.g., U.S. Pat. No. 6,333,195.
  • MMV Murine Leukemia Virus
  • Mink-Cell focus- inducing Virus e.g., Murine Sarcoma Virus, Reticuloendotheliosis virus, Gibbon Abe Leukemia Virus, Mason Pfizer Monkey Virus, or Rous Sarcoma Virus, see, e.g., U.S. Pat. No. 6,333,195.
  • the recombinant retrovirus is a lentivirus (e.g., Human
  • HIV-l Immunodeficiency Virus-l
  • SIV Simian Immunodeficiency Virus
  • FV Feline Immunodeficiency Virus
  • the recombinant retrovirus may comprise a viral polypeptide (e.g., retroviral env) to aid entry into the target cell.
  • a viral polypeptide e.g., retroviral env
  • retroviral env e.g., retroviral env
  • the viral polypeptide may be an amphotropic viral polypeptide, e.g., amphotropic env, which aids entry into cells derived from multiple species, including cells outside of the original host species.
  • the viral polypeptide may be a xenotropic viral polypeptide that aids entry into cells outside of the original host species.
  • the viral polypeptide is an ecotropic viral polypeptide, e.g., ecotropic env, which aids entry into cells of the original host species.
  • viral polypeptides capable of aiding entry of retroviruses into cells include but are not limited to: MMLV amphotropic env, MMLV ecotropic env, MMLV xenotropic env, vesicular stomatitis virus-g protein (VSV-g), HIV-l env, Gibbon Ape Leukemia Virus (GALV) env, RD114, FeLV-C, FeLV-B, MLV 10A1 env gene, and variants thereof, including chimeras. See e.g., Yee et al, (1994), Methods Cell Biol., Pt A:99-l l2 (VSV-G); U.S. Pat. No. 5,449,614.
  • the viral polypeptide is genetically modified to promote expression or enhanced binding to a receptor.
  • a recombinant virus is produced by introducing a viral DNA or
  • RNA construct into a producer cell In some cases, the producer cell does not express exogenous genes. In other cases, the producer cell is a“packaging cell” comprising one or more exogenous genes, e.g., genes encoding one or more gag, pol, or env polypeptides and/or one or more retroviral gag, pol, or env polypeptides.
  • the retroviral packaging cell may comprise a gene encoding a viral polypeptide, e.g., VSV-g that aids entry into target cells.
  • the packaging cell comprises genes encoding one or more lentiviral proteins, e.g., gag, pol, env, vpr, vpu, vpx, vif, tat, rev, or nef.
  • the packaging cell comprises genes encoding adenovirus proteins such as E1A or E1B or other adenoviral proteins.
  • proteins supplied by packaging cells may be retrovirus-derived proteins such as gag, pol, and env; lentivirus-derived proteins such as gag, pol, env, vpr, vpu, vpx, vif, tat, rev, and nef; and adenovirus-derived proteins such as E1A and E1B.
  • the packaging cells supply proteins derived from a virus that differs from the virus from which the viral vector derives.
  • Packaging cell lines include but are not limited to any easily -transfectable cell line. Packaging cell lines can be based on 293T cells, NIH3T3, COS or HeLa cell lines.
  • Packaging cells are often used to package virus vector plasmids deficient in at least one gene encoding a protein required for virus packaging. Any cells that can supply a protein or polypeptide lacking from the proteins encoded by such virus vector plasmid may be used as packaging cells.
  • packaging cell lines include but are not limited to: Platinum-E (Plat-E); Platinum-A (Plat-A); BOSC 23 (ATCC CRL 11554); and Bing (ATCC CRL 11270), see, e.g., Morita et al, (2000), Gene Therapy, 7: 1063-1066; Onishi et al., (1996), Experimental Hematology, 24:324-329; and U.S. Pat. No.
  • the retroviral construct may be derived from a range of retroviruses, e.g., MMLV, HIV-l, SIV, FIV, or other retrovirus described herein.
  • the retroviral construct may encode all viral polypeptides necessary for more than one cycle of replication of a specific virus. In some cases, the efficiency of viral entry is improved by the addition of other factors or other viral polypeptides. In other cases, the viral polypeptides encoded by the retroviral construct do not support more than one cycle of replication, e.g., U.S. Pat. No. 6,872,528. In such circumstances, the addition of other factors or other viral polypeptides can help facilitate viral entry.
  • the recombinant retrovirus is HIV-l virus comprising a VSV-g polypeptide but not comprising a HIV-l env polypeptide.
  • the retroviral construct may comprise: a promoter, a multi-cloning site, and/or a resistance gene.
  • promoters include but are not limited to CMV, SV40, EFla, ⁇ -actin; retroviral LTR promoters, and inducible promoters.
  • the retroviral construct may also comprise a packaging signal (e.g., a packaging signal derived from the MFG vector; a psi packaging signal).
  • a packaging signal e.g., a packaging signal derived from the MFG vector; a psi packaging signal.
  • examples of some retroviral constructs known in the art include but are not limited to: pMX, pBabeX or derivatives thereof. See e.g., Onishi et al. (1996) Experimental Hematology 24:324- 329.
  • the retroviral construct is a self-inactivating lentiviral vector (SIN) vector, see, e.g., Miyoshi et al. (1998) J. Virol. 72(10):8150-8157.
  • the retroviral construct is LL-CG, LS-CG, CL-CG, CS-CG, CLG or MFG. Miyoshi et al. (1998) J. Virol. 72(10):8150-8157; Onishi et al. (1996) Experimental Hematology 24:324-329; Riviere et al. (1995) Proc. Natl. Acad. Sci. USA 92:6733- 6737.
  • Virus vector plasmids include: pMXs, pMxs-IB, pMXs-puro, pMXs-neo (pMXs-IB is a vector carrying the blasticidin-resistant gene instead of the puromycin-resistant gene of pMXs-puro) Kimatura et al. (2003) Experimental Hematology 31: 1007-1014; MFG Riviere et al. (1995) Proc. Natl. Acad. Sci. USA 92:6733-6737; pBabePuro; Morgenstem et al. (1990) Nucleic Acids Research 18:3587-3596; LL-CG, CL-CG, CS-CG, CLG Miyoshi et al. (1998) J. Virol.
  • the retroviral construct comprises blasticidin (e.g., pMXs-IB), puromycin (e.g., pMXs-puro, pBabePuro); or neomycin (e.g., pMXs- neo).
  • blasticidin e.g., pMXs-IB
  • puromycin e.g., pMXs-puro, pBabePuro
  • neomycin e.g., pMXs- neo
  • a nucleic acid construct can be introduced into a cell using a variety of well- known techniques, such as non-viral based transfection of the cell.
  • the construct is incorporated into a vector and introduced into a host cell.
  • Introduction into the cell may be performed by any non-viral based transfection known in the art, such as, but not limited to, electroporation, calcium phosphate mediated transfer, nucleofection, sonoporation, heat shock, magnetofection, liposome mediated transfer, microinjection, microprojectile mediated transfer (nanoparticles), cationic polymer mediated transfer (DEAE-dextran, polyethylenimine, polyethylene glycol (PEG) and the like) or cell fusion.
  • electroporation calcium phosphate mediated transfer
  • nucleofection sonoporation
  • heat shock magnetofection
  • liposome mediated transfer microinjection
  • microprojectile mediated transfer nanoprojectile mediated transfer (nanoparticles)
  • transfection reagents such as LipofectamineTM, Dojindo HilymaxTM, FugeneTM, jetPEITM, EffecteneTM, and DreamFectTM.
  • a suitable gene editing system e.g. a site specific gene editing system such as a programmable gene editing system
  • a gene editing system as described herein may make use of a “targeted nuclease”.
  • a“targeted nuclease” it is meant a nuclease that cleaves a specific DNA sequence to produce a double strand break at that sequence.
  • a TALEN system which includes a transcription activator-like effector (TALE) protein fused to a nuclease (e.g., Fokl nuclease) - TALEN, or suitable vector and/or vectors encoding same, for targeted integration of a nucleic acid sequence encoding an OTX2 polypeptide, a nucleic acid sequence encoding a PAX6 polypeptide, and/or a nucleic acid sequence encoding a MITF polypeptide.
  • TALE transcription activator-like effector
  • a recombinant vector according to the present disclosure may serve as a donor vector for use with a suitable TALEN system designed to introduce a nucleic acid sequence encoding an OTX2 polypeptide, a nucleic acid sequence encoding a PAX6 polypeptide, and/or a nucleic acid sequence encoding a MITF polypeptide, e.g., an OTX2-T2A-PAX6- T2A-MITF encoding nucleic acid, into the genome of an iPSC.
  • a suitable TALEN system designed to introduce a nucleic acid sequence encoding an OTX2 polypeptide, a nucleic acid sequence encoding a PAX6 polypeptide, and/or a nucleic acid sequence encoding a MITF polypeptide, e.g., an OTX2-T2A-PAX6- T2A-MITF encoding nucleic acid, into the genome of an iPSC.
  • Such a donor vector may include an expression cassette including an inducible promoter, e.g., a Tet-0 promoter, driving expression of OTX2-T2A-PAX6-T2A-MITF.
  • a suitable TALEN system may include, e.g., suitable left and right TALEN plasmids.
  • the TALEN system is specifically configured to integrate a nucleic acid sequence encoding an OTX2 polypeptide, a nucleic acid sequence encoding a PAX6 polypeptide, and/or a nucleic acid sequence encoding a MITF polypeptide, e.g., an OTX2-T2A-PAX6-T2A-MITF encoding nucleic acid, at the AAVS1 locus and/or the CLYBL locus of an iPSC genome.
  • TALENs are described in greater detail in US Patent Application No. 2011/0145940, the disclosure of which is incorporated by reference herein. See also, Christian M. et al. Nucleic Acids Res. 2011
  • a targeted nuclease that finds use in the subject methods is a zinc finger nuclease or“ZFN”, which may include, e.g., a designed zinc finger domain and/or a selected zinc finger domain.
  • ZFNs are targeted nucleases comprising a nuclease fused to a zinc finger DNA binding domain (see, e.g., DeKelver et al, Genome Research 2010).
  • ZFBD it is meant a polypeptide domain that binds DNA in a sequence-specific manner through one or more zinc fingers.
  • a zinc finger is a domain of about 30 amino acids within the zinc finger binding domain whose structure is stabilized through coordination of a zinc ion.
  • Examples of zinc fingers include C2H2 zinc fingers, C3H zinc fingers, and C4 zinc fingers.
  • A“designed” zinc finger domain is a domain not occurring in nature whose design/composition results principally from rational criteria, e.g., application of substitution rules and computerized algorithms for processing information in a database storing information of existing ZFP designs and binding data. See, for example, U.S. Pat. Nos. 6,140,081; 6,453,242; and 6,534,261; see also WO 98/53058; WO 98/53059; WO 98/53060; and WO
  • A“selected” zinc finger domain is a domain not found in nature whose production results primarily from an empirical process such as phage display, interaction trap or hybrid selection.
  • ZFNs are described in greater detail in US Patent No. 7,888,121 and US Patent No. 7,972,854, the disclosures of which are incorporated herein by reference.
  • the most recognized example of a ZFN in the art is a fusion of the Fokl nuclease with a zinc finger DNA binding domain.
  • targeted nucleases include naturally occurring and recombinant nucleases, e.g. CRISPR/Cas9, restriction endonucleases, meganucleases homing endonucleases, and the like. See, e.g., F. Oceguera-Yanez et al. Methods. 101 (2016) 43-55, which discusses the adaptation of TALEN and CRISPR/Cas9 nuclease technologies in order to introduce transgenes into the AAVS1 locus in iPSCs.
  • targeted nucleases are used in pairs, with one targeted nuclease specific for one sequence of an integration site and the second targeted nuclease specific for a second sequence of an integration site.
  • any targeted nuclease(s) that are specific for the integration site of interest and promote the cleavage of an integration site may be used.
  • the targeted nuclease(s) may be stably expressed by the cells.
  • the targeted nuclease(s) may be transiently expressed by the cells, e.g. it may be provided to the cells prior to, simultaneously with, or subsequent to contacting the cells with the donor vector.
  • the targeted nuclease(s) may be provided to cells as DNA, e.g. plasmid or vector, as described herein, e.g., by using transfection, nucleofection, or the like.
  • targeted nuclease(s) may be provided to cells as mRNA encoding the targeted nuclease(s), e.g. using well-developed transfection techniques; see, e.g. Angel and Yanik (2010) PLoS ONE 5(7): el 1756; Beumer et al.
  • the targeted nuclease(s) may be provided to cells as a polypeptide.
  • Such polypeptides may optionally be fused to a polypeptide domain that increases solubility of the product, and/or fused to a polypeptide permeant domain to promote uptake by the cell.
  • the targeted nuclease(s) may be produced by eukaryotic cells or by prokaryotic cells, it may be further processed by unfolding, e.g. heat denaturation, DTT reduction, etc. and may be further refolded, using methods known in the art. It may be modified, e.g. by chemical derivatization or by molecular biology techniques and synthetic chemistry, e.g. so as to improve resistance to proteolytic degradation or to optimize solubility properties.
  • the present disclosure provides a recombinant vector, e.g., a donor vector for use with a suitable gene editing system, including a promoter, a nucleic acid including a nucleotide sequence encoding an OTX2 polypeptide operably linked to the promoter, a nucleic acid including a nucleotide sequence encoding a PAX6 polypeptide operably linked to the promoter; and a nucleic acid including a nucleotide sequence encoding an MITF polypeptide operably linked to the promoter.
  • a recombinant vector e.g., a donor vector for use with a suitable gene editing system, including a promoter, a nucleic acid including a nucleotide sequence encoding an OTX2 polypeptide operably linked to the promoter, a nucleic acid including a nucleotide sequence encoding a PAX6 polypeptide operably linked to the promoter; and a nucleic acid including
  • the present disclosure provides a recombinant vector, e.g., a donor vector for use with a suitable gene editing system, including an inducible promoter, e.g., a Tet-inducible promoter, an exogenous nucleic acid including a nucleotide sequence encoding an OTX2 polypeptide, an exogenous nucleic acid including a nucleotide sequence encoding a PAX6 polypeptide, and an exogenous nucleic acid including a nucleotide sequence encoding an MITF polypeptide, wherein one or more of the nucleotide sequence encoding an OTX2 polypeptide, the nucleotide sequence encoding a PAX6 polypeptide, and the nucleotide sequence encoding an MITF polypeptide are operably linked to the inducible promoter.
  • an inducible promoter e.g., a Tet-inducible promoter
  • the present disclosure provides a recombinant vector, e.g., a donor vector for use with a suitable gene editing system, including an exogenous nucleic acid comprising a nucleotide sequence encoding an OTX2 polypeptide, an exogenous nucleic acid including a nucleotide sequence encoding a PAX6 polypeptide, and an exogenous nucleic acid including a nucleotide sequence encoding an MITF polypeptide, wherein the recombinant vector is targeted to an AAVS1 locus or a CLYBL locus of an iPSC.
  • a recombinant vector e.g., a donor vector for use with a suitable gene editing system, including an exogenous nucleic acid comprising a nucleotide sequence encoding an OTX2 polypeptide, an exogenous nucleic acid including a nucleotide sequence encoding a PAX6 polypeptide, and an exogenous nucleic
  • a variety of suitable promoters are known in the art, which may be used in connection with the recombinant vectors described herein, including for example, ubiquitous promoters (CMV, CAG, CBA, EFla) and tissue specific promoters (Synapsin, CamKIIa, GFAP, RPE, ALB, TBG, MBP, MCK, TNT, and aMHC).
  • the promoter may be an inducible promoter, such as a Tet-inducible promoter, e.g., a PTRE3G or PTRE3GS promoter. See, e.g., Zhou et al. Gene Ther. 2006 Oct;l3(l9): l382- 90. Epub 2006 May 25; and Heinz et al. (2011) Human Gene Ther. 22(2): 166-176.
  • polypeptide encodes a biologically active OTX2 polypeptide having an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 95%, 97%, or at least 99% amino acid sequence identity, or any other percent identity from at least 70% to 100%, to the OTX2 amino acid sequence depicted in Figure 12 or to the amino acid sequence provided by one of
  • the nucleotide sequence encoding a PAX6 polypeptide encodes a biologically active PAX6 polypeptide having an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 95%, 97%, or at least 99% amino acid sequence identity, or any other percent identity from at least 70% to 100%, to the PAX6 amino acid sequence depicted in Figure 13 or to the amino acid sequence provided by one of
  • NP_00l245393 l
  • NP_00l245394.1 NP_00l297087.
  • l NP_001297088.1
  • the nucleotide sequence encoding a MITF polypeptide encodes a biologically active MITF polypeptide having an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 95%, 97%, or at least 99% amino acid sequence identity, or any other percent identity from at least 70% to 100%, to the MITF amino acid sequence depicted in Figure 14 or to the amino acid sequence provided by one of
  • NP_000239.1 NP_00l 171896.1 , NP_00l 171897.1, ⁇ _001341533.1,
  • NP_001341534.1 ⁇ _001341535.1
  • ⁇ _001341536.1 ⁇ _001341537.1
  • NP_006713.1 NP_93780l. l, NP_937802. l, NP_937820. l, and NP_93782l.2.
  • the recombinant vector may also express one or more reporter molecules, e.g., one or more fluorescent reporters, e.g., GFP, RFP, mCherry, CFP, YFP, tdTomato, etc.).
  • the vector does not include a nucleotide sequence encoding a transcription factor other than an OTX2 polypeptide, a PAX6 polypeptide, and/or an MITF polypeptide.
  • the recombinant vector does not include a tyrosinase enhancer.
  • Recombinant vectors according to the present disclosure may exclude a nucleic acid that encodes any one or more of the following: TFEC, LHX2, VMD2, CFTR, RPE65, MFRP, CTRP5, CFH, C3, C2B, APOE, APOB, mTOR, FOXO, AMPK, SIRT1-6, HTRP1, ABCA4, TIMP3, VEGFA, CFI, TLR3, TLR4, APP, CD46, BACE1, ELOVL4, ADAM 10, CD55, CD59, and ARMS2.
  • nucleotide sequence encoding an OTX2 is a nucleotide sequence encoding an OTX2
  • polypeptide is separated from the nucleotide sequence encoding a PAX6 polypeptide by a first ribosomal skipping sequence, and the nucleotide sequence encoding a PAX6 polypeptide is separated from the nucleotide sequence encoding a MITF polypeptide by a second ribosomal skipping sequence.
  • Ribosomal skipping sequences are known in the art, including, e.g., sequences encoding the T2A (EGRGS LLTCGD VEENP GP (SEQ ID NO:6), P2A (ATNF SLLKQ AGDVEENPGP (SEQ ID NO: 7), E2A (QCTNYALLKLAGDVESNPGP (SEQ ID NO: 8)) and F2A (VKQTLNFDLLKLAGDVESNPGP (SEQ ID NO:9) peptides. (GSG) residues can be added to the 5' end of the peptide to improve cleavage efficiency.
  • the nucleotide sequence encoding an OTX2 polypeptide is position closer to the promoter, e.g., the Tet-inducible promoter, than the nucleotide sequence encoding a PAX6 polypeptide, and the nucleotide sequence encoding a PAX6 polypeptide is positioned closer to the promoter than the nucleotide sequence encoding a MITF polypeptide.
  • the above vectors may be used to genetically modify one or more host cells.
  • the present disclosure provides a genetically modified host cell, e.g., a genetically modified iPSC, including an exogenous nucleic acid including a nucleotide sequence encoding an OTX2 polypeptide, an exogenous nucleic acid including a nucleotide sequence encoding a PAX6 polypeptide, and an exogenous nucleic acid including a nucleotide sequence encoding an MITF polypeptide.
  • a genetically modified host cell e.g., a genetically modified iPSC, including an exogenous nucleic acid including a nucleotide sequence encoding an OTX2 polypeptide, an exogenous nucleic acid including a nucleotide sequence encoding a PAX6 polypeptide, and an exogenous nucleic acid including a nucleotide sequence encoding an MITF polypeptide.
  • the exogenous nucleic acid including a nucleotide sequence encoding an OTX2 polypeptide, the exogenous nucleic acid including a nucleotide sequence encoding a PAX6 polypeptide, and the exogenous nucleic acid including a nucleotide sequence encoding an MITF polypeptide are integrated into the genome of the genetically modified host cell, e.g., a genetically modified iPSC.
  • the exogenous nucleic acids are maintained in an episomal state.
  • the host cell may also include one or more gene editing tools as described herein, e.g. a TALEN, a ZFN, one or more nucleic acids encoding a TALEN, and/or one or more nucleic acids encoding a ZFN.
  • the genetically modified host cell does not include an exogenous nucleic acid(s) encoding a transcription factor other than an OTX2 polypeptide, a PAX6 polypeptide, and/or an MITF polypeptide.
  • the genetically modified host cell does not include an exogenous tyrosinase enhancer.
  • the genetically modified host cell does not include an exogenous nucleic acid(s) encoding one or more of the following: TFEC, LHX2, VMD2, CFTR, RPE65, MFRP, CTRP5, CFH, C3, C2B, APOE, APOB, mTOR, FOXO, AMPK, SIRT1-6, HTRP1, ABCA4, TIMP3, VEGFA, CFI, TLR3, TLR4, APP, CD46, BACE1, ELOVL4, ADAM 10, CD55, CD59, and ARMS2.
  • exogenous nucleic acid(s) encoding one or more of the following: TFEC, LHX2, VMD2, CFTR, RPE65, MFRP, CTRP5, CFH, C3, C2B, APOE, APOB, mTOR, FOXO, AMPK, SIRT1-6, HTRP1, ABCA4, TIMP3, VEGFA, CFI, TLR3, TLR4, APP, CD46, BACE
  • IPCs Induced pluripotent stem cells
  • iPSCs cells for use in the disclosed methods may be obtained from any source.
  • suitable source e.g., a commercial provider, or prepared from any suitable somatic cell of interest.
  • suitable somatic cell of interest e.g., any cells other than germ cells of mammalian origin (such as, humans, non-human primates, (e.g., monkeys), pigs, rodents (e.g., mice or rats), etc.) can be used as starting material for the production of iPSCs.
  • Examples include keratinizing epithelial cells, mucosal epithelial cells, exocrine gland epithelial cells, endocrine cells, liver cells, epithelial cells, endothelial cells, fibroblasts, muscle cells, cells of the blood and the immune system, cells of the nervous system including nerve cells and glia cells, pigment cells, and progenitor cells, including hematopoietic stem cells, amongst others.
  • the degree of cell differentiation the age of an animal from which cells are collected and the like; even undifferentiated progenitor cells (including somatic stem cells) and finally differentiated mature cells can be used as sources of somatic cells in connection with the preparation of iPSCs.
  • the somatic cell is itself a RPE cell such as a human RPE cell.
  • the RPE cell can be an adult or a fetal RPE cell.
  • the somatic cell is a fetal human RPE cell, obtained from a human fetus of about week 9 to about week 38 of gestation, such as about week 9 to about week 16 of gestation, about week 17 to about week 25 of gestation, about 16 to about 19 weeks, or about week 26 to about week 38 of gestation.
  • the somatic cell includes at least one allele encoding a mutation associated with an RPE-associated disease, e.g. a mutation in the RPE65 gene and/or the BEST1 gene.
  • somatic cells are not particularly limited. Allogeneic cells can be used.
  • the iPSCs are not matched for MHC (e.g., HLA).
  • the somatic cells are obtained from an individual, for example to provide patient-specific RPE. This may be desirable, for example, where the RPE that is ultimately generated will be used to test specific active agents or specific genome editing tools for use in a particular patient or subgroup of patients.
  • Patient-specific RPE generated according to a method as described herein may also be transplanted into a patient, e.g., a patient from which the somatic cells used to prepare the iPSCs were derived, e.g., to treat a disease characterized by degeneration of RPE, e.g., age-related macular degeneration (AMD), macular dystrophies such as Stargardf s and Stargardf s-like disease, Best disease (vitelliform macular dystrophy), and adult vitelliform dystrophy or subtypes of retinitis pigmentosa.
  • AMD age-related macular degeneration
  • macular dystrophies such as Stargardf s and Stargardf s-like disease
  • Best disease vitelliform macular dystrophy
  • adult vitelliform dystrophy or subtypes of retinitis pigmentosa may take place following genomic modification of the RPE cells, e.g., using a genome editing tool as described herein.
  • Somatic cells isolated from a mammal can be pre-cultured using a medium known to be suitable for their cultivation according to the choice of cells before being subjected to the step of nuclear reprogramming.
  • a medium known to be suitable for their cultivation include, but are not limited to, minimal essential medium (MEM) containing about 5 to 20% fetal calf serum (FCS), Dulbecco’s modified Eagle medium (DMEM), RPMI 1640 medium, 199 medium, F12 medium, and the like.
  • MEM minimal essential medium
  • FCS fetal calf serum
  • DMEM Dulbecco’s modified Eagle medium
  • RPMI 1640 medium 199 medium
  • F12 medium and the like.
  • the medium can exclude ingredients derived from non-human animals, such as FCS.
  • Media comprising a basal medium supplemented with human-derived ingredients suitable for cultivation of various somatic cells (particularly, recombinant human proteins such as growth factors), non-essential amino acids, vitamins and the like are commercially available; those skilled in the art are able to choose an appropriate xeno-free medium according to the source of somatic cells. Somatic cells pre-cultured using a xeno-free medium are dissociated from the culture vessel using an appropriate xeno-free cell dissociation solution, and recovered, after which they are brought into contact with nuclear reprogramming substances.
  • cells are cultured at about 35 to 38° C, usually at 37° C, in about 4- 6% CO2 generally at 5% CO2, unless specifically indicated otherwise below.
  • Somatic cells can be reprogrammed to produce induced pluripotent stem cells (iPSCs) using methods known to one of skill in the art.
  • iPSCs induced pluripotent stem cells
  • One of skill in the art can readily produce induced pluripotent stem cells, see for example, U.S. Patent
  • nuclear reprogramming factors are used to produce pluripotent stem cells from a somatic cell.
  • at least three, or at least four, of Klf4, c-Myc, Oct3/4, Sox2, Nanog, and Lin28 are utilized.
  • Oct3/4, Sox2, c-Myc and Klf4 is utilized.
  • the somatic cells are treated with a nuclear reprogramming substance, which is generally one or more factor(s) capable of inducing an iPSC from a somatic cell or a nucleic acid that encodes these substances (including forms integrated in a vector).
  • the nuclear reprogramming substances generally include at least Oct3/4, Klf4 and Sox2 or nucleic acids that encode these molecules.
  • a functional inhibitor of p53, L- myc or a nucleic acid that encodes L-myc, and Lin28 or Lin28b or a nucleic acid that encodes Lin28 or Lin28b, can be utilized as additional nuclear reprogramming substances.
  • Nanog can also be utilized for nuclear reprogramming. As disclosed in U.S. Patent Application Publication No.
  • exemplary reprogramming factors for the production of iPSCs include (1) Oct3/4, Klf4, Sox2, L-Myc (Sox2 can be replaced with Soxl, Sox3, Soxl5, Soxl7 or Soxl8; Klf4 is replaceable with Klfl, Klf2 or Klf5); (2) Oct3/4, Klf4, Sox2, L-Myc, TERT, SV40 Large T antigen
  • Klf4, Sox2, L-Myc, SV40LT (11) Oct3/4, Esrrb, Sox2, L-Myc (Esrrb is replaceable with Esrrg); (12) Oct3/4, Klf4, Sox2; (13) Oct3/4, Klf4, Sox2, TERT, SV40LT; (14) Oct3/4, Klf4, Sox2, TERT, HPV16 E6; (15) Oct3/4, Klf4, Sox2, TERT, HPV16 E7; (16) Oct3/4, Klf4, Sox2, TERT, HPV16 E6, HPV16 E7; (17) Oct3/4, Klf4, Sox2, TERT, Bmil; (18) Oct3/4, Klf4, Sox2, Lin28 (19) Oct3/4, Klf4, Sox2, Lin28, SV40LT; (20) Oct3/4, Klf4, Sox2, Lin28, TERT, SV40LT; (21) Oct3/4, Klf4, Sox2, SV40LT; or (22) Oct3/4, Esrrb, So
  • Oct3/4, Klf4, Sox2, and c-Myc are utilized.
  • Oct4, Nanog, and Sox2 are utilized, see for example, U.S. Pat. No. 7,682,828, the disclosure of which is incorporated by reference herein.
  • These factors include, but are not limited to, Oct3/4, Klf4 and Sox2.
  • the factors include, but are not limited to Oct 3/4, Klf4 and Myc.
  • Oct3/4, Klf4, c-Myc, and Sox2 are utilized.
  • Oct3/4, Klf4, Sox2 and Sal 4 are utilized.
  • the cell After being cultured with nuclear reprogramming substances, the cell can, for example, be cultured under conditions suitable for culturing ES cells.
  • the culture In the case of mouse cells, the culture is carried out with the addition of Leukemia Inhibitory Factor (LIF) as a differentiation suppression factor to an ordinary medium.
  • LIF Leukemia Inhibitory Factor
  • bFGF basic fibroblast growth factor
  • the cell is cultured in the co-presence of mouse
  • Mouse embryonic fibroblasts in common use as feeders include the STO cell line (ATCC CRL-1503) and the like; for induction of an iPSC, useful cells can be generated by stably integrating the neomycin resistance gene and the LIF gene in the STO cell (SNL76/7 STO cell; ECACC 07032801) (McMahon, A. P. & Bradley, A. Cell 62, 1073-1085, 1990) and the like can be used. Mitomycin C- treated MEFs are commercially available from Millipore. Gamma-irradiated MEFs are commercially available from Global Stem Cells. Generally, somatic cells are transduced with reprogramming factors in the absence of MEFs. In some aspects of Mitomycin C- treated MEFs are commercially available from Millipore. Gamma-irradiated MEFs are commercially available from Global Stem Cells. Generally, somatic cells are transduced with reprogramming factors in the absence of MEFs.
  • the cells are re-seeded onto MEFs.
  • the iPSC can be modified to express one or more exogenous nucleic acids.
  • non-RPE cells present in a mixed cell population comprising the RPE cell and other, non-RPE cells, where possible non-RPE cells include host iPSC cells that failed to differentiate into RPE cells.
  • an RPE cell will be purified from a mixed cell
  • an RPE cell can be sorted based on phenotypic features characteristic of RPE cells. Sorting can involve contacting each cell with an antibody or ligand that binds to a marker characteristic of an RPE cell, followed by separation of the specifically recognized cells from other cells in the population.
  • Sorting can involve contacting each cell with an antibody or ligand that binds to a marker characteristic of an RPE cell, followed by separation of the specifically recognized cells from other cells in the population.
  • One method is immunopanning, in which specific antibody is coupled to a solid surface. The cells are contacted with the surface, and cells not expressing the marker are washed away. The bound cells are then recovered by more vigorous elution. Variations of this are affinity chromatography and antibody- mediated magnetic cell sorting.
  • the cells are contacted with a specific primary antibody, and then captured with a secondary antiimmunoglobulin reagent bound to a magnetic bead.
  • the adherent cells are then recovered by collecting the beads in a magnetic field.
  • cells selected positively for expression of BEST1 can provide a population that is 60%, 70%, 80%, 90%, or greater than 90% (e.g., 95%, 98%, 99%, or greater than 99%), BEST1 positive.
  • cells selected positively for expression of RPE65 can provide a population that is 60%, 70%, 80%, 90%, or greater than 90% (e.g., 95%, 98%, 99%, or greater than 99%), RPE65 positive.
  • RPE cell population comprising RPE cells and non-RPE cells results in a purified RPE cell population, where at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or more than 99%, of the cells of the purified RPE cell population are RPE cells.
  • a subject RPE cell can be amplified, e.g., to obtain a purified RPE cell
  • RPE cells comprising from about 10 3 to about 10 8 or more purified RPE cells, e.g., from about 10 3 purified RPE cells to about 10 4 purified induced RPE cells, from about 10 4 purified RPE cells to about 10 5 purified RPE cells, from about 10 5 purified RPE cells to about 10 6 purified RPE cells, from about 10 6 purified RPE cells to about 10 7 purified RPE cells, from about 10 7 purified RPE cells to about 10 8 purified RPE cells, or more than 10 8 purified RPE cells.
  • purified RPE cells e.g., from about 10 3 purified RPE cells to about 10 4 purified induced RPE cells, from about 10 4 purified RPE cells to about 10 5 purified RPE cells, from about 10 5 purified RPE cells to about 10 6 purified RPE cells, from about 10 6 purified RPE cells to about 10 7 purified RPE cells, from about 10 7 purified RPE cells to about 10 8 purified RPE cells, or more than 10 8 purified RPE
  • a subject RPE cell (or a population of purified RPE cells) can be stored.
  • a subject RPE cell, or a population of purified RPE cells can be cryopreserved, using well-established methods. See for example, PCT Publication No. WO 2012/149484 A2, the disclosure of which is incorporated by reference herein.
  • the cells can be cryopreserved with or without a substrate.
  • the storage temperature ranges from about -50° C. to about -60° C, about -600° C.
  • cryopreserved cells lower temperatures are used for the storage (e.g., maintenance) of the cryopreserved cells.
  • liquid nitrogen or other similar liquid coolant
  • the cells are stored for greater than about 6 hours.
  • the cells are stored about 72 hours.
  • the cells are stored 48 hours to about one week.
  • the cells are stored for about 1, 2, 3, 4, 5, 6, 7, or 8 weeks.
  • the cells are stored for 1, 2, 3, 4, 5, 67, 8, 9, 10, 11 or 12 months.
  • the cells can also be stored for longer times.
  • the cells can be cryopreserved separately or on a substrate, such as any of the substrates disclosed herein.
  • a general method of cry opreserving cells is disclosed herein, that can be used for cryopreservation of any cell type, such as stem cells, including iPSCs, and differentiated cells, such as RPE cells.
  • the method includes the use of alginate.
  • Alginate (or“alginic acid” or“algin”) refers to the anionic polysaccharide distributed widely in the cell walls of brown algae. Alginate forms water-soluble salts with alkali metals, such as sodium, potassium, lithium, magnesium, ammonium, and the substituted ammonium cations derived from lower amines, such as methyl amine, ethanol amine, diethanol amine, and triethanol amine.
  • Alginate includes calcium alginate, sodium alginate, propylene-glycol alginate, and potassium alginate.
  • cells such as the disclosed RPE cells are contacted with an effective amount of alginate.
  • the cells are contacted with alginate, and then exposed to divalent cations, such as Calcium, Barium, Copper, Zinc or Strontium) which results in cross-linking of the alginate polymers in the cell/liquid alginate suspension (see for example, U.S. Patent Application Publication No. 20120171295, the disclosure of which is incorporated by reference herein).
  • divalent cations such as Calcium, Barium, Copper, Zinc or Strontium
  • the divalent cation used to cross-link the alginate in the cell/liquid alginate solution is calcium chloride (CaCh), barium chloride (BaCf), strontium chloride (SrCh), copper chloride (CuCh), or zinc chloride (ZnCh).
  • the divalent cation used to cross-link the alginate in the cell/liquid alginate solution is calcium chloride (CaCL).
  • the solution of divalent cation comprises about 0.5%, about 0.75%, about 1.0%, about 1.25%, about 1.5%, about 1.75%, or about 2.0% divalent cation.
  • the solution of divalent cation comprises 1.5% divalent cation, e.g., CaCh.
  • additional cryoprotectants can be used.
  • the cells can be cryopreserved in a cryopreservation solution comprising one or more cryoprotectants, such as DMSO, serum albumin, such as human or bovine serum albumin.
  • the solution comprises about 1%, about 1.5%, about 2%, about 2.5%, about 3%, about 4%, about 5%, about 6%) about 7%, about 8%, about 9%, or about 10% DMSO.
  • the solution comprises about 1% to about 3%, about 2% to about 4%, about 3% to about 5%, about 4% to about 6%, about 5% to about 7%, about 6% to about 8%, about 7% to about 9%, or about 8% to about 10% DMSO or albumin.
  • the solution comprises 2,5% DMSO, In another specific embodiment, the solution comprises 10% DMSO.
  • Cells can be cryopreserved in small containers (e.g., ampoules); in bags suitable for cryopreservation; or in any other suitable container for cryopreservation.
  • cells are cryopreserved in commercially available cryopreservation medium.
  • the cells can be cryopreserved in a cryopreservation solution comprising one or more solutions for use in storing cells.
  • Cryopreservation solutions included CryoStor CS10® and HYPOTHERMOSOl® (BioLife Solutions, Bothell, Wash.)).
  • the solution comprises about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, or about 70% HYPOTHERMOSOL®. In other embodiments, the solution comprises about 25% to about 50%, about 40% to about 60%, about 50% to about 60%, about 50% to about 70%, or about 60% to about 70% HYPOTHERMOSOL®. In a specific embodiment, the solution comprises 55% HYPOTHERMOSOL®. In another specific embodiment, the solution comprises 57.5% HYPOTHERMOSOL®.
  • the cryopreservation solution can include one or more excipients, such as dextran, starch, glucose, lactose, sucrose, gelatin, silica gel, glycerol monostearate, sodium chloride, glycerol, propylene, and/or glycol.
  • the cryopreservation solution can include media, such as the media disclosed herein.
  • the medium can be phosphate buffered saline or Dulbecco's Modified Eagle's Medium (DMEM).
  • Cells may be cooled, for example, at about 1° C/minute during
  • cryopreservation In some embodiments, the cryopreservation temperature is about - 80° C to about -180° C, or about -125° C to about -140° C. In some embodiments, the cells are cooled to 4° C prior to cooling at about 1° C/minute. Cryopreserved cells can be transferred to vapor phase of liquid nitrogen prior to thawing for use. In some embodiments, for example, once the cells have reached about -80° C, they are transferred to a liquid nitrogen storage area. Cryopreservation can also be done using a controlled-rate freezer. Cryopreserved cells may be thawed, e.g., at a temperature of about 25° C to about 40° C, and typically at a temperature of about 37° C.
  • compositions comprising RPE cells
  • the present disclosure provides a composition comprising a subject RPE cell (or a population of RPE cells, e.g., a population of purified RPE cells, e.g., such as a population of purified RPE cells in an RPE monolayer).
  • a subject composition can include various components in addition to the RPE cells.
  • a subject composition can include a subject RPE cell(s) and a culture medium.
  • the culture medium comprises one or more growth factors.
  • the culture medium is a serum-free culture medium.
  • the composition comprises RPE cells and a cryopreservative agent, e.g., a cryopreservation medium, e.g. as described previously herein.
  • a cryopreservative agent e.g., a cryopreservation medium, e.g. as described previously herein.
  • a subject composition can include a subject RPE cell(s) and a matrix, e.g., a matrix component.
  • Suitable matrix components include, e.g., collagen; gelatin;
  • fibrin ; fibrinogen; laminin; a glycosaminoglycan; elastin; hyaluronic acid; a proteoglycan; a glycan; poly(lactic acid); poly(vinyl alcohol); poly(vinyl
  • the matrix comprises one or more of collagen, gelatin, fibrin, fibrinogen, laminin, and elastin; and can further comprise a non-proteinaceous polymer, e.g., can further comprise one or more of poly(lactic acid), poly(vinyl alcohol), poly(vinyl pyrrobdone), poly(ethylene oxide), poly(caprolactone), poly(hydroxy butyric acid), cellulose, a cellulose derivative, starch, and a starch derivative.
  • the matrix comprises one or more of collagen, gelatin, fibrin, fibrinogen, laminin, and elastin; and can further comprise hyaluronic acid, a proteoglycan, a glycosaminoglycan, or a glycan.
  • the matrix comprises collagen
  • the collagen can comprise type I collagen, type II collagen, type III collagen, type V collagen, type XI collagen, and combinations thereof.
  • the matrix can be a hydrogel.
  • a suitable hydrogel is a polymer of two or more monomers, e.g., a homopolymer or a heteropolymer comprising multiple monomers.
  • Suitable hydrogel monomers include the following: lactic acid, glycolic acid, acrylic acid, 1 -hydroxy ethyl methacrylate (HEMA), ethyl methacrylate (EMA), propylene glycol methacrylate (PEMA), acrylamide (AAM), N-vinylpyrrolidone, methyl methacrylate (MMA), glycidyl methacrylate (GDMA), glycol methacrylate (GMA), ethylene glycol, fumaric acid, and the like.
  • Common cross linking agents include tetraethylene glycol dimethacrylate (TEGDMA) and ⁇ , ⁇ '- methylenebisacrylamide.
  • the hydrogel can be homopolymeric, or can comprise copolymers of two or more of the aforementioned polymers.
  • exemplary hydrogels include, but are not limited to, a copolymer of poly(ethylene oxide) (PEO) and polypropylene oxide) (PPO); PluronicTM F-127 (a difunctional block copolymer of PEO and PPO of the nominal formula EO100-PO65-EO100, where EO is ethylene oxide and PO is propylene oxide); poloxamer 407 (a tri-block copolymer consisting of a central block of polypropylene glycol) flanked by two hydrophilic blocks of polypthylene glycol)); a polypthylene oxide)-poly(propylene oxide)-poly(ethylene oxide) co-polymer with a nominal molecular weight of 12,500 Daltons and a PEO:PPO ratio of 2: 1); a poly(/V-isopropylacrylamide)-base hydrogel (a PNIPAAm-
  • the cell density in a subject RPE cell composition can range from about 10 2 cells/mm 3 to about 10 9 cells/mm 3 , e.g., from about 10 2 cells/mm 3 to about 10 4 cells/mm 3 , from about 10 4 cells/mm 3 to about 10 6 cells/mm 3 , from about 10 6 cells/mm 3 to about 10 7 cells/mm 3 , from about 10 7 cells/mm 3 to about 10 8 cells/mm 3 , or from about 10 8 cells/mm 3 to about 10 9 cells/mm 3 .
  • the matrix can take any of a variety of forms, or can be relatively amorphous.
  • the matrix can be in the form of a sheet, a cylinder, a sphere, etc.
  • a subject RPE, RPE cell, or population of RPE cells is useful in various aspects
  • methods are provided for identifying an agent that alters the proliferation of RPE cells.
  • the methods include contacting an RPE cell with an effective amount of an agent of interest.
  • methods are provided for identifying an agent that increases differentiation of RPE.
  • methods are provided for identifying an agent that affects RPE cell survival, and/or changes the endogenous expression of genes in RPE cells.
  • Therapeutic agents can be identified for the treatment of disease using these methods.
  • methods are provided for identifying an agent that affects the epithelial phenotype of RPE cells. The method includes contacting iPSCs, RPE or RPE cells with the agent of interest, and assaying the production and/or survival and/or phenotype of RPE cells.
  • the agent can be introduced into any step of the methods disclosed herein.
  • a screening method which includes contacting RPE or an RPE cell or cells as described herein, or generated using a method as described herein, with a test agent, and assaying to determine the effect of the test agent on the RPE or RPE cell or cells, e.g., assaying to determine the effect of the test agent on differentiation and/or proliferation and/or production and/or survival and/or phenotype of the RPE or RPE cell or cells.
  • RPE cells are treated with a stressor, such as thapsigargin, A23187, DL-dithiotreitol, or 2- deoxy-D-glucose.
  • a stressor such as thapsigargin, A23187, DL-dithiotreitol, or 2- deoxy-D-glucose.
  • RNA or protein expression (RNA or protein) of one or more of the following genes may be assessed: MITF (GENBANK® Accession No. NM-000248), PAX6 (GENBANK® Accession No. NM-000280), LHX2 (GENBANK® Accession No. NM-004789), TFEC (GENBANK® Accession No. NM— 012252), CDH1 (GENBANK® Accession No. NM-004360), CDH3 (GENBANK® Accession No. NM-001793), CLDN10 (GENBANK® Accession No. NM— 182848), CLDN16 (GENBANK® Accession No.
  • NM-006580 CLDN19 (GENBANK® Accession No. NM-148960), BEST1 (GENBANK® Accession No. NM— 004183), TIMP3 (GENBANK® Accession No. NM-000362), TRPM1 (GENBANK® Accession No. NM-002420), TRPM3 (GENBANK® Accession No. NM— 020952), TTR (GENBANK® Accession No. NM-000371), VEGFA
  • Assessment of the activity of candidate pharmaceutical compounds generally involves combining a subject RPE, a subject RPE cell, or a subject population of RPE cells with the candidate compound, either alone or in combination with other drugs.
  • the investigator determines any change in the morphology, marker phenotype, or functional activity of the cells that is attributable to the compound (compared with untreated cells or cells treated with an inert compound), and then correlates the effect of the compound with the observed change.
  • Cytotoxicity can be determined in the first instance by the effect on cell
  • Effect of cell function can be assessed using any standard assay to observe phenotype or activity of RPE, an RPE cell, or population of RPE cells, such as assaying for epithelial integrity of RPE cells by measuring trans epithelial resistance (TER) and/or assaying RPE cells for the ability to phagocytose rod cell OS at a rate similar to immortalized RPE cells.
  • TER trans epithelial resistance
  • a subject screening method generally includes appropriate controls, e.g., a control sample that lacks the test agent.
  • appropriate controls e.g., a control sample that lacks the test agent.
  • a plurality of assay mixtures is run in parallel with different agent concentrations to obtain a differential response to the various concentrations.
  • one of these concentrations serves as a negative control, i.e. at zero concentration or below the level of detection.
  • a variety of other reagents may be included in the screening assay. These include reagents such as salts, neutral proteins, e.g. albumin, detergents, etc that are used to facilitate optimal protein-protein binding and/or reduce non-specific or background interactions. Reagents that improve the efficiency of the assay, such as protease inhibitors, nuclease inhibitors, anti-microbial agents, etc. may be used.
  • the components of the assay mixture are added in any order that provides for the requisite binding or other activity. Incubations are performed at any suitable temperature, typically between 4°C and 40°C. Incubation periods are selected for optimum activity, but may also be optimized to facilitate rapid high-throughput screening. Typically, between 0.1 and 1 hour will be sufficient.
  • Candidate agents encompass numerous chemical classes, typically synthetic, semi-synthetic, or naturally occurring inorganic or organic molecules.
  • Candidate agents include those found in large libraries of synthetic or natural compounds.
  • synthetic compound libraries are commercially available from Maybridge Chemical Co. (Trevillet, Cornwall, UK), ComGenex (South San Francisco, CA), and MicroSource (New Milford, CT).
  • a rare chemical library is available from Aldrich (Milwaukee, Wis.) and can also be used.
  • libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are available from Pan Labs (Bothell, WA) or are readily producible.
  • Candidate agents may be small organic or inorganic compounds having a molecular weight of more than 50 and less than about 2,500 daltons.
  • Candidate agents may comprise functional groups necessary for structural interaction with proteins, e.g., hydrogen bonding, and may include at least an amine, carbonyl, hydroxyl or carboxyl group, and may contain at least two of the functional chemical groups.
  • the candidate agents may comprise cyclical carbon or heterocyclic structures and/or aromatic or poly aromatic structures substituted with one or more of the above functional groups.
  • Candidate agents are also found among biomolecules including peptides, saccharides, fatty acids, steroids, purines, pyrimidines, derivatives, structural analogs or combinations thereof.
  • a test agent can be a small molecule.
  • the test molecules may be individual small molecules of choice or in some cases, the small molecule test agents to be screened come from a combinatorial library, i.e., a collection of diverse chemical compounds generated by either chemical synthesis or biological synthesis by combining a number of chemical“building blocks.”
  • a linear combinatorial chemical library such as a polypeptide library is formed by combining a set of chemical building blocks called amino acids in every possible way for a given compound length (i.e., the number of amino acids in a polypeptide compound). Millions of chemical compounds can be synthesized through such combinatorial mixing of chemical building blocks.
  • Combinatorial chemical libraries include, but are not limited to: diversomers such as hydantoins, benzodiazepines, and dipeptides, as described in, e.g., Hobbs et al, (1993), Proc. Natl. Acad. Sci. U.S.A., 90:6909-6913; analogous organic syntheses of small compound libraries, as described in Chen et al, (1994), J. Amer. Chem. Soc., 116:2661-2662; Oligocarbamates, as described in Cho, et al, (1993), Science, 261 : 1303-1305; peptidyl phosphonates, as described in Campbell et al, (1994), J. Org.
  • combinatorial libraries are commercially available from, e.g., ComGenex (Princeton, N.J.); Asinex (Moscow, Russia); Tripos, Inc. (St. Louis,
  • a test agent is a gene editing system or a component thereof, as described in greater detail below.
  • a subject RPE, RPE cell, or population of RPE cells which finds use in the disclosed screening methods may be an RPE, RPE cell, or population of RPE, which was differentiated using a method as described herein from an iPSC derived from a cell from an individual with a family history or genetic make-up of interest for an RPE related disease, e.g., age-related macular degeneration.
  • a subject RPE, RPE cell, or population of RPE cells finds use in methods designed to screen one or more gene editing tools (i.e., a component of a gene editing system, e.g., a site specific gene editing system such as a programmable gene editing system).
  • a subject RPE, RPE cell, or population of RPE cells is a patient- derived RPE, RPE cell, or population of RPE cells, e.g., which include a genetic defect, such as a mutant allele (dominant negative) of the BEST1 gene.
  • a subject RPE, RPE cell, or population of RPE cells may be contacted with (i) a CRISPR/Cas guide RNA, (ii) a DNA encoding a CRISPR/Cas guide RNA, (iii) a DNA and/or RNA encoding a programmable gene editing protein such as a zinc finger protein (ZFP) (e.g., a zinc finger nuclease - ZFN), a transcription activator- like effector (TALE) protein (e.g., fused to a nuclease - TALEN), a DNA-guided polypeptide such as Natronobacterium gregoryi Argonaute (NgAgo), and/or a CRISPR/Cas RNA-guided polypeptide (e.g., Cas9, CasX, CasY, Cpfl, and the like); (iv) a DNA donor template; (v) a nucleic acid molecule (DNA,
  • a DNA encoding a resolvase and/or invertase e.g., Gin, Hin, ⁇ 3, Tn3, Sin, Beta, and the like
  • a transposon and/or a DNA derived from a transposon e.g., bacterial transposons such as Tn3, Tn5, Tn7, Tn9, TnlO, Tn903, Tnl68l, and the like;
  • eukaryotic transposons such as Tcl/mariner super family transposons, PiggyBac superfamily transposons, hAT superfamily transposons, PiggyBac, Sleeping Beauty, Frog Prince, Minos, Himarl, and the like).
  • a gene editing tool includes a protein, e.g., a gene editing protein such as a ZFP (e.g., ZFN), a TALE (e.g., TALEN), a DNA-guided polypeptide such as Natronobacterium gregoryi Argonaute (NgAgo), a CRISPR/Cas RNA-guided polypeptide (e.g., Cas9, CasX, CasY, Cpfl, and the like), a site-specific recombinase (e.g., Cre recombinase, Dre recombinase, Flp recombinase, KD recombinase, B2 recombinase, B3 recombinase, R
  • a gene editing protein such as a ZFP (e.g., ZFN), a TALE (e.g., TALEN), a DNA-guided polypeptide such as Natronobacterium
  • a recombinase e.g., Gin, Hin, ⁇ 3, Tn3, Sin, Beta, and the like
  • a transposase e.g., a transposase related to transposons such as bacterial transposons such as Tn3, Tn5, Tn7, Tn9, TnlO, Tn903, Tnl68l, and the like; or eukaryotic transposons such as Tcl/mariner super family transposons, PiggyBac superfamily transposons, hAT superfamily transposons, PiggyBac, Sleeping Beauty, Frog Prince, Minos, Himarl, and
  • a site-specific gene editing system such as a programmable gene editing system
  • can include a single component e.g., a ZFP, a ZFN, a TALE, a TALEN, a site-specific recombinase, a resolvase / integrase, a transpose, a transposon, and the like
  • a gene editing system includes at least two components.
  • a gene editing system e.g.
  • a programmable gene editing system includes (i) a donor template nucleic acid; and (ii) a gene editing protein (e.g., a programmable gene editing protein such as a ZFP, a ZFN, a TALE, a TALEN, a DNA-guided polypeptide such as Natronobacterium gregoryi Argonaute (NgAgo), a CRISPR/Cas RNA-guided polypeptide such as Cas9, CasX, CasY, or Cpfl, and the like), or a nucleic acid molecule encoding the gene editing protein (e.g., DNA or RNA such as a plasmid or mRNA).
  • a gene editing protein e.g., a programmable gene editing protein such as a ZFP, a ZFN, a TALE, a TALEN, a DNA-guided polypeptide such as Natronobacterium gregoryi Argonaute (NgAgo), a
  • a gene editing system (e.g. a programmable gene editing system) includes (i) a CRISPR/Cas guide RNA, or a DNA encoding the CRISPR/Cas guide RNA; and (ii) a CRISPR/CAS RNA-guided polypeptide (e.g., Cas9, CasX, CasY, Cpfl, and the like), or a nucleic acid molecule encoding the RNA-guided polypeptide (e.g., DNA or RNA such as a plasmid or mRNA).
  • a gene editing system e.g.
  • a programmable gene editing system includes (i) an NgAgo-like guide DNA; and (ii) a DNA-guided polypeptide (e.g., NgAgo), or a nucleic acid molecule encoding the DNA-guided polypeptide (e.g., DNA or RNA such as a plasmid or mRNA).
  • a gene editing system e.g.
  • a programmable gene editing system includes at least three components: (i) a donor DNA template; (ii) a CRISPR/Cas guide RNA, or a DNA encoding the CRISPR/Cas guide RNA; and (iii) a CRISPR/Cas RNA-guided polypeptide (e.g., Cas9, CasX, CasY, or Cpfl), or a nucleic acid molecule encoding the RNA-guided polypeptide (e.g., DNA or RNA such as a plasmid or mRNA).
  • a gene editing system e.g.
  • a programmable gene editing system includes at least three components: (i) a donor DNA template; (ii) an NgAgo-like guide DNA, or a DNA encoding the NgAgo-like guide DNA; and (iii) a DNA-guided polypeptide (e.g., NgAgo), or a nucleic acid molecule encoding the DNA-guided polypeptide (e.g., DNA or RNA such as a plasmid or mRNA).
  • a donor DNA template includes at least three components: (i) a donor DNA template; (ii) an NgAgo-like guide DNA, or a DNA encoding the NgAgo-like guide DNA; and (iii) a DNA-guided polypeptide (e.g., NgAgo), or a nucleic acid molecule encoding the DNA-guided polypeptide (e.g., DNA or RNA such as a plasmid or mRNA).
  • a subject RPE, RPE cell or population of RPE cells is
  • a subject RPE, RPE cell or population of RPE cells is contacted with one or more components of a gene editing system (i.e., one or more gene editing tools).
  • a target cell might already include one of the components of a gene editing system and the user need only add the remaining components.
  • the a subject RPE, RPE cell or population of RPE cells does not necessarily need to be contacted with all of the components of a given gene editing system.
  • a target RPE cell might already include a gene editing protein (e.g., a ZFP, a TALE, a DNA-guided polypeptide (e.g., NgAgo), a CRISPR/Cas RNA-guided polypeptide such Cas9, CasX, CasY, Cpfl, and the like, a site-specific recombinase such as Cre recombinase, Dre recombinase, Flp recombinase, KD recombinase, B2 recombinase, B3 recombinase, R recombinase, Hin recombinase, Tre recombinase, PhiC3l integrase, Bxbl integrase, R4 integrase, lambda integrase, HK022 integrase, HP1 integrase
  • a gene editing protein e.g.
  • the target RPE cell cell may already include a CRISPR/Cas guide RNA and/or a DNA encoding the guide RNA or an NgAgo-like guide DNA, and the RPE cell can be contacted with one or more of: (i) a donor template; and (ii) a CRISPR/Cas RNA- guided polypeptide (e.g., Cas9, CasX, CasY, Cpfl, and the like), or a nucleic acid molecule encoding the RNA-guided polypeptide (e.g., DNA or RNA such as a plasmid or mRNA); or a DNA-guided polypeptide (e.g., NgAgo), or a nucleic acid molecule encoding the DNA-guided polypeptide.
  • a CRISPR/Cas guide RNA e.g., Cas9, CasX, CasY, Cpfl, and the like
  • a nucleic acid molecule encoding the RNA
  • a gene editing system need not be a system that‘edits’ a nucleic acid.
  • a gene editing system can be used to modify target nucleic acids (e.g., DNA and/or RNA) in a variety of ways without creating a double strand break (DSB) in the target DNA.
  • target nucleic acids e.g., DNA and/or RNA
  • a double stranded target DNA is nicked (one strand is cleaved), and in some cases (e.g., in some cases where the gene editing protein is devoid of nuclease activity, e.g., a CRISPR/Cas RNA-guided polypeptide may harbor mutations in the catalytic nuclease domains), the target nucleic acid is not cleaved at all.
  • a CRISPR/Cas protein e.g., Cas9, CasX, CasY, Cpfl
  • a heterologous protein domain is fused to a heterologous protein domain.
  • the heterologous protein domain can provide an activity to the fusion protein such as (i) a DNA-modifying activity (e.g., nuclease activity, methyltransferase activity, demethylase activity, DNA repair activity, DNA damage activity, deamination activity, dismutase activity, alkylation activity, depurination activity, oxidation activity, pyrimidine dimer forming activity, integrase activity, transposase activity, recombinase activity, polymerase activity, ligase activity, helicase activity, photolyase activity or glycosylase activity), (ii) a transcription modulation activity (e.g., fusion to a transcriptional repressor or activator), or (iii) an activity that modifies a protein (e.g., a histone) that is associated with target DNA (e.g., methyltransferase activity, demethylase activity,
  • a DNA-modifying activity e.g., nuclea
  • a gene editing system can be used in applications that modify a target nucleic acid in way that do not cleave the target nucleic acid, and can also be used in applications that modulate transcription from a target DNA.
  • CRISPR/Cas RNa-guided proteins such as Cas9, CasX, CasY, and Cpfl
  • Zinc finger proteins such as Zinc finger nucleases
  • TALE proteins such as TALENs
  • CRISPR/Cas guide RNAs refer to, for example, Dreier, et al, (2001) J Biol Chem 276:29466-78; Dreier, et al, (2000) J Mol Biol 303:489-502; Liu, et al, (2002) J Biol Chem 277:3850-6); Dreier, et al., (2005) J Biol Chem 280:35588-97; Jamieson, et al, (2003) Nature Rev Drug Discov 2:361-8; Durai, et al, (2005) Nucleic Acids Res 33:5978-90; Segal, (2002) Methods 26:76-83; Porteus and Carroll, (2005 ) Nat Biotechnol 23:967-73; Pabo, et al, (2001) Ann Rev Biochem 70:313-40; Wolfe, et al, (2000) Ann Rev Biophys Biomol Struct 29: 183-212; Segal and Barbas, (2001) Curr Op
  • a subject RPE, RPE cell, or population of RPE cells finds use in methods of treating a disease characterized by degeneration of RPE, e.g., age-related macular degeneration (AMD), macular dystrophies such as Stargardf s and Stargardf s-like disease, Best disease (vitelliform macular dystrophy), and adult vitelliform dystrophy or subtypes of retinitis pigmentosa.
  • AMD age-related macular degeneration
  • macular dystrophies such as Stargardf s and Stargardf s-like disease, Best disease (vitelliform macular dystrophy), and adult vitelliform dystrophy or subtypes of retinitis pigmentosa.
  • Such treatment may take the form of transplantation, e.g., subretinal transplantation, of RPE prepared according to a method as described herein.
  • transplantation may take place following genomic modification of the RPE cells, e.g., using a genome editing tool as described herein.
  • a subject RPE, RPE cell, or population of RPE cells is a patient-derived RPE, RPE cell, or population of RPE cells, e.g., which include a genetic defect, such as a mutant allele (dominant negative) of the BEST1 gene.
  • Patient-specific RPE generated according to a method as described herein may be transplanted, e.g., via subretinal transplantation, into a patient, e.g., a patient from which the somatic cells used to prepare the iPSCs were derived, e.g., to treat a disease characterized by degeneration of RPE as described above.
  • RPE retinal pigment epithelium
  • iPSCs induced pluripotent stem cells
  • an exogenous nucleic acid comprising a nucleotide sequence encoding an OTX2 polypeptide, an exogenous nucleic acid comprising a nucleotide sequence encoding a PAX6 polypeptide, and an exogenous nucleic acid comprising a nucleotide sequence encoding an MITF polypeptide to provide genetically modified iPSCs;
  • the method does not comprise introducing into the iPSCs or genetically modified iPSCs a transcription factor other than an OTX2 polypeptide, a PAX6 polypeptide, and/or an MITF polypeptide, or an exogenous nucleic acid comprising a nucleotide sequence encoding a transcription factor other than an OTX2 polypeptide, a PAX6 polypeptide, and/or an MITF polypeptide; thereby generating RPE.
  • exogenous nucleic acid comprising a nucleotide sequence encoding an OTX2 polypeptide
  • exogenous nucleic acid comprising a nucleotide sequence encoding an MITF polypeptide are comprised by a recombinant vector.
  • the gene editing system comprises a gene editing tool selected from a TALEN, a ZFN, one or more nucleic acids encoding a TALEN, and one or more nucleic acids encoding a ZFN.
  • the method of 5, comprising introducing into the iPSCs or genetically modified iPSCs one or more nucleic acids encoding a TALEN or a nucleic acid encoding a ZFN.
  • iPSCs are human iPSCs.
  • iPSCs are rodent iPSCs.
  • iPSCs are non-human primate iPSCs.
  • OTX2 polypeptide comprises an amino acid sequence having at least about 80% amino acid identity to the amino acid sequence set forth in SEQ ID NO: 13.
  • PAX6 polypeptide comprises an amino acid sequence having at least about 80% amino acid identity to the amino acid sequence set forth in SEQ ID NO: 17.
  • MITF polypeptide comprises an amino acid sequence having at least about 80% amino acid identity to the amino acid sequence set forth in SEQ ID NO: 19.
  • the method of 14, wherein the culturing the genetically modified iPSCs under conditions sufficient for expression of the OTX2 polypeptide, the PAX6 polypeptide, and the MITF polypeptide comprises contacting the genetically modified iPSCs with tetracycline or a derivative or analogue thereof to induce expression of the OTX2 polypeptide, the PAX6 polypeptide, and the MITF polypeptide.
  • the method of any one of 1-16 comprising exposing the RPE to conditions sufficient for termination of expression of the OTX2 polypeptide, the PAX6 polypeptide, and the MITF polypeptide from the exogenous nucleic acids.
  • any one of 2-19 wherein, in the recombinant vector, the nucleotide sequence encoding an OTX2 polypeptide is separated from the nucleotide sequence encoding a PAX6 polypeptide by a first ribosomal skipping sequence, and the nucleotide sequence encoding a PAX6 polypeptide is separated from the nucleotide sequence encoding a MITF polypeptide by a second ribosomal skipping sequence.
  • the recombinant vector comprises a promoter and each of the nucleotide sequence encoding an OTX2 polypeptide, the nucleotide sequence encoding a PAX6 polypeptide, and the nucleotide sequence encoding a MITF polypeptide is operably linked to the promoter.
  • the method of any one of 2-22 comprising culturing the genetically modified iPSCs under conditions sufficient for replication of the genetically modified iPSCs prior to the culturing the genetically modified iPSCs under conditions sufficient for expression of the OTX2 polypeptide, the PAX6 polypeptide, and the MITF polypeptide, and differentiation of the genetically modified iPSCs into retinal pigment epithelial cells.
  • an exogenous nucleic acid comprising a nucleotide sequence encoding an OTX2 polypeptide, an exogenous nucleic acid comprising a nucleotide sequence encoding a PAX6 polypeptide, and an exogenous nucleic acid comprising a nucleotide sequence encoding an MITF polypeptide to provide genetically modified iPSCs;
  • the method does not comprise introducing into the iPSCs or genetically modified iPSCs an exogenous nucleic acid comprising a tyrosinase enhancer; thereby generating RPE.
  • exogenous nucleic acid comprising a nucleotide sequence encoding an OTX2 polypeptide
  • exogenous nucleic acid comprising a nucleotide sequence encoding an MITF polypeptide are comprised by a recombinant vector.
  • the gene editing system comprises a gene editing tool selected from a TALEN, a ZFN, one or more nucleic acids encoding a TALEN, and one or more nucleic acids encoding a ZFN.
  • the method of 30, comprising introducing into the iPSCs or genetically modified iPSCs one or more nucleic acids encoding a TALEN or a nucleic acid encoding a ZFN.
  • iPSCs are human iPSCs.
  • iPSCs are rodent iPSCs.
  • iPSCs are non-human primate iPSCs.
  • OTX2 polypeptide comprises an amino acid sequence having at least about 80% amino acid identity to the amino acid sequence set forth in SEQ ID NO: 13.
  • PAX6 polypeptide comprises an amino acid sequence having at least about 80% amino acid identity to the amino acid sequence set forth in SEQ ID NO: 17.
  • MITF polypeptide comprises an amino acid sequence having at least about 80% amino acid identity to the amino acid sequence set forth in SEQ ID NO: 19.
  • the method of 38, wherein the culturing the genetically modified iPSCs under conditions sufficient for expression of the OTX2 polypeptide, the PAX6 polypeptide, and the MITF polypeptide comprises contacting the genetically modified iPSCs with tetracycline or a derivative or analogue thereof to induce expression of the OTX2 polypeptide, the PAX6 polypeptide, and the MITF polypeptide.
  • the method of any one of 26-40 comprising exposing the RPE to conditions sufficient for termination of expression of the OTX2 polypeptide, the PAX6 polypeptide, and the MITF polypeptide from the exogenous nucleic acids.
  • any one of 27-43 wherein, in the recombinant vector, the nucleotide sequence encoding an OTX2 polypeptide is separated from the nucleotide sequence encoding a PAX6 polypeptide by a first ribosomal skipping sequence, and the nucleotide sequence encoding a PAX6 polypeptide is separated from the nucleotide sequence encoding a MITF polypeptide by a second ribosomal skipping sequence.
  • the method of 44 wherein the first and/or second ribosomal skipping sequence encodes a 2A peptide selected from T2A, P2A, E2A, and F2A.
  • the recombinant vector comprises a promoter and each of the nucleotide sequence encoding an OTX2 polypeptide, the nucleotide sequence encoding a PAX6 polypeptide, and the nucleotide sequence encoding a MITF polypeptide is operably linked to the promoter.
  • any one of 27-46 comprising culturing the genetically modified iPSCs under conditions sufficient for replication of the genetically modified iPSCs prior to the culturing the genetically modified iPSCs under conditions sufficient for expression of the OTX2 polypeptide, the PAX6 polypeptide, and the MITF polypeptide, and differentiation of the genetically modified iPSCs into retinal pigment epithelial cells.
  • a recombinant vector comprising a Tet-inducible promoter, an exogenous nucleic acid comprising a nucleotide sequence encoding an OTX2 polypeptide, an exogenous nucleic acid comprising a nucleotide sequence encoding a PAX6 polypeptide, and an exogenous nucleic acid comprising a nucleotide sequence encoding an MITF polypeptide to provide genetically modified iPSCs, wherein one or more of the nucleotide sequence encoding an OTX2 polypeptide, the nucleotide sequence encoding a PAX6 polypeptide, and the nucleotide sequence encoding an MITF polypeptide are operably linked to the Tet- inducible promoter; and
  • the recombinant vector is a donor vector for a gene editing system.
  • the gene editing system comprises a gene editing tool selected from a TALEN, a ZFN, one or more nucleic acids encoding a TALEN, and one or more nucleic acids encoding a ZFN.
  • the method of 52 comprising introducing into the iPSCs or genetically modified iPSCs one or more nucleic acids encoding a TALEN or a nucleic acid encoding a ZFN.
  • iPSCs are human iPSCs.
  • iPSCs are rodent iPSCs.
  • iPSCs are non-human primate iPSCs.
  • OTX2 polypeptide comprises an amino acid sequence having at least about 80% amino acid identity to the amino acid sequence set forth in SEQ ID NO: 13.
  • PAX6 polypeptide comprises an amino acid sequence having at least about 80% amino acid identity to the amino acid sequence set forth in SEQ ID NO: 17.
  • MITF polypeptide comprises an amino acid sequence having at least about 80% amino acid identity to the amino acid sequence set forth in SEQ ID NO: 19.
  • the method of any one of 49-59, wherein the culturing the genetically modified iPSCs under conditions sufficient for expression of the OTX2 polypeptide, the PAX6 polypeptide, and the MITF polypeptide comprises contacting the genetically modified iPSCs with tetracycline or a derivative or analogue thereof to induce expression of the OTX2 polypeptide, the PAX6 polypeptide, and the MITF polypeptide.
  • any one of 49-61 comprising exposing the RPE to conditions sufficient for termination of expression of the OTX2 polypeptide, the PAX6 polypeptide, and the MITF polypeptide from the exogenous nucleic acids.
  • any one of 49-64 wherein, in the recombinant vector, the nucleotide sequence encoding an OTX2 polypeptide is separated from the nucleotide sequence encoding a PAX6 polypeptide by a first ribosomal skipping sequence, and the nucleotide sequence encoding a PAX6 polypeptide is separated from the nucleotide sequence encoding a MITF polypeptide by a second ribosomal skipping sequence.
  • the method of 65 wherein the first and/or second ribosomal skipping sequence encodes a 2A peptide selected from T2A, P2A, E2A, and F2A.
  • any one of 49-66 comprising culturing the genetically modified iPSCs under conditions sufficient for replication of the genetically modified iPSCs prior to the culturing the genetically modified iPSCs under conditions sufficient for expression of the OTX2 polypeptide, the PAX6 polypeptide, and the MITF polypeptide, and differentiation of the genetically modified iPSCs into retinal pigment epithelial cells.
  • any one of 49-67 wherein the vector is configured such that expression level of the OTX2 polypeptide is greater than expression level of the PAX6 polypeptide, and expression level of the PAX6 polypeptide is greater than expression level of the MITF polypeptide.
  • a recombinant vector comprising an exogenous nucleic acid comprising a nucleotide sequence encoding an OTX2 polypeptide, an exogenous nucleic acid comprising a nucleotide sequence encoding a PAX6 polypeptide, and an exogenous nucleic acid comprising a nucleotide sequence encoding an MITF polypeptide to provide genetically modified iPSCs, wherein the recombinant vector is targeted to an AAVS1 locus or a CLYBL locus of the iPSCs; and
  • the recombinant vector is a donor vector for a gene editing system.
  • the gene editing system comprises a gene editing tool selected from a TALEN, a ZFN, one or more nucleic acids encoding a TALEN, and one or more nucleic acids encoding a ZFN.
  • the method of 72 comprising introducing into the iPSCs or genetically modified iPSCs one or more nucleic acids encoding a TALEN or a nucleic acid encoding a ZFN.
  • iPSCs are rodent iPSCs.
  • iPSCs are non-human primate iPSCs.
  • OTX2 polypeptide comprises an amino acid sequence having at least about 80% amino acid identity to the amino acid sequence set forth in SEQ ID NO: 13.
  • PAX6 polypeptide comprises an amino acid sequence having at least about 80% amino acid identity to the amino acid sequence set forth in SEQ ID NO: 17.
  • MITF polypeptide comprises an amino acid sequence having at least about 80% amino acid identity to the amino acid sequence set forth in SEQ ID NO: 19.
  • any one of 69-79 comprising exposing the RPE to conditions sufficient for termination of expression of the OTX2 polypeptide, the PAX6 polypeptide, and the MITF polypeptide from the exogenous nucleic acids.
  • any one of 69-81 wherein, in the recombinant vector, the nucleotide sequence encoding an OTX2 polypeptide is separated from the nucleotide sequence encoding a PAX6 polypeptide by a first ribosomal skipping sequence, and the nucleotide sequence encoding a PAX6 polypeptide is separated from the nucleotide sequence encoding a MITF polypeptide by a second ribosomal skipping sequence.
  • the method of 82 wherein the first and/or second ribosomal skipping sequence encodes a 2A peptide selected from T2A, P2A, E2A, and F2A.
  • any one of 69-83 comprising culturing the genetically modified iPSCs under conditions sufficient for replication of the genetically modified iPSCs prior to the culturing the genetically modified iPSCs under conditions sufficient for expression of the OTX2 polypeptide, the PAX6 polypeptide, and the MITF polypeptide, and differentiation of the genetically modified iPSCs into retinal pigment epithelial cells.
  • any one of 69-84 wherein the vector is configured such that expression level of the OTX2 polypeptide is greater than expression level of the PAX6 polypeptide, and expression level of the PAX6 polypeptide is greater than expression level of the MITF polypeptide.
  • a recombinant vector comprising a promoter, an exogenous nucleic acid comprising a nucleotide sequence encoding an OTX2 polypeptide, an exogenous nucleic acid comprising a nucleotide sequence encoding a PAX6 polypeptide, and an exogenous nucleic acid comprising a nucleotide sequence encoding an MITF polypeptide to provide genetically modified iPSCs, wherein each of the nucleotide sequence encoding an OTX2 polypeptide, the nucleotide sequence encoding a PAX6 polypeptide, and the nucleotide sequence encoding an MITF polypeptide is operably linked to the promoter; and
  • the method of 86, wherein the recombinant vector is a donor vector for a gene editing system.
  • the gene editing system comprises a gene editing tool selected from a TALEN, a ZFN, one or more nucleic acids encoding a TALEN, and one or more nucleic acids encoding a ZFN.
  • the method of 89 comprising introducing into the iPSCs or genetically modified iPSCs one or more nucleic acids encoding a TALEN or a nucleic acid encoding a ZFN.
  • iPSCs are human iPSCs.
  • iPSCs are rodent iPSCs.
  • any one of 86-88, wherein the iPSCs are non-human primate iPSCs.
  • MITF polypeptide comprises an amino acid sequence having at least about 80% amino acid identity to the amino acid sequence set forth in SEQ ID NO: 19.
  • any one of 86-96 comprising exposing the RPE to conditions sufficient for termination of expression of the OTX2 polypeptide, the PAX6 polypeptide, and the MITF polypeptide from the exogenous nucleic acids.
  • any one of 86-98 wherein, in the recombinant vector, the nucleotide sequence encoding an OTX2 polypeptide is separated from the nucleotide sequence encoding a PAX6 polypeptide by a first ribosomal skipping sequence, and the nucleotide sequence encoding a PAX6 polypeptide is separated from the nucleotide sequence encoding a MITF polypeptide by a second ribosomal skipping sequence..
  • the method of 99 wherein the first and/or second ribosomal skipping sequence encodes a 2A peptide selected from T2A, P2A, E2A, and F2A.
  • any one of 86-100 comprising culturing the genetically modified iPSCs under conditions sufficient for replication of the genetically modified iPSCs prior to the culturing the genetically modified iPSCs under conditions sufficient for expression of the OTX2 polypeptide, the PAX6 polypeptide, and the MITF polypeptide, and differentiation of the genetically modified iPSCs into retinal pigment epithelial cells.
  • the method of any one of 1-102 comprising culturing the genetically modified iPSCs for a period of time under differentiation conditions prior to culturing the genetically modified iPSCs under conditions sufficient for expression of the OTX2 polypeptide, the PAX6 polypeptide, and the MITF polypeptide in the genetically modified iPSCs. .
  • the method of 103 wherein the period of time is from about 2 to about 4 days.
  • the method of 104, wherein the differentiation conditions comprise culturing the genetically modified iPSCs in a differentiation medium.
  • the method of 105, wherein the differentiation medium is a neural induction medium.
  • a composition comprising the retinal pigment epithelium (RPE) of 107.
  • RPE retinal pigment epithelium
  • composition of 108 wherein the composition comprises a
  • a screening method comprising:
  • RPE retinal pigment epithelium
  • the screening method of 111 wherein the method is a multiplexed screening method.
  • the screening method of 111 or 112, wherein the assaying comprises assaying for modulation of retinal pigment epithelial cell proliferation and/or modulation of retinal pigment epithelial cell differentiation.
  • the screening method of 113 wherein the modulation is increased retinal pigment epithelial cell proliferation and/or increased retinal pigment epithelial cell differentiation relative to retinal pigment epithelial cell proliferation and/or retinal pigment epithelial cell differentiation in the absence of the test agent.
  • the screening method of 113 wherein the modulation is decreased retinal pigment epithelial cell proliferation and/or decreased retinal pigment epithelial cell differentiation relative to retinal pigment epithelial cell proliferation and/or retinal pigment epithelial cell differentiation in the absence of the test agent.
  • a kit comprising the retinal pigment epithelium (RPE) of 107 or the composition of one of 108-110 and a liquid culture medium.
  • the kit of 117 comprising one or more of fetal bovine serum, an epithelial cell growth supplement, and an antibiotic.
  • a recombinant vector comprising:
  • nucleic acid comprising a nucleotide sequence encoding an OTX2 polypeptide operably linked to the promoter
  • nucleic acid comprising a nucleotide sequence encoding a PAX6 polypeptide operably linked to the promoter
  • nucleic acid comprising a nucleotide sequence encoding an MITF polypeptide operably linked to the promoter.
  • the recombinant vector of 119 wherein the vector does not comprise a nucleotide sequence encoding a transcription factor other than an OTX2 polypeptide, a PAX6 polypeptide, and/or an MITF polypeptide.
  • the recombinant vector of 122 wherein the recombinant vector is a donor vector for a gene editing system.
  • the recombinant vector of 123 wherein the gene editing system comprises a gene editing tool selected from a TALEN, a ZFN, one or more nucleic acids encoding a TALEN, and one or more nucleic acids encoding a ZFN.
  • OTX2 polypeptide comprises an amino acid sequence having at least about 80% amino acid identity to the amino acid sequence set forth in SEQ ID NO: 13.
  • the recombinant vector of 129 wherein the mammalian cell is selected from a human cell, a rodent cell, and a non-human primate cell.
  • the recombinant vector of any one of 119- 131 wherein the nucleotide sequence encoding an OTX2 polypeptide is separated from the nucleotide sequence encoding a PAX6 polypeptide by a first ribosomal skipping sequence, and the nucleotide sequence encoding a PAX6 polypeptide is separated from the nucleotide sequence encoding a MITF polypeptide by a second ribosomal skipping sequence.
  • the recombinant vector of 132 wherein the first and/or second ribosomal skipping sequence encodes a 2A peptide selected from T2A, P2A, E2A, and F2A.
  • a recombinant vector comprising:
  • an exogenous nucleic acid comprising a nucleotide sequence encoding an OTX2 polypeptide
  • an exogenous nucleic acid comprising a nucleotide sequence encoding a PAX6 polypeptide
  • an exogenous nucleic acid comprising a nucleotide sequence encoding an MITF polypeptide, wherein one or more of the nucleotide sequence encoding an OTX2 polypeptide, the nucleotide sequence encoding a PAX6 polypeptide, and the nucleotide sequence encoding an MITF polypeptide are operably linked to the inducible promoter.
  • the recombinant vector of 134 wherein the vector does not comprise a nucleotide sequence encoding a transcription factor other than an OTX2 polypeptide, a PAX6 polypeptide, and/or an MITF polypeptide.
  • the recombinant vector of 138 wherein the gene editing system comprises a gene editing tool selected from a TALEN, a ZFN, one or more nucleic acids encoding a TALEN, and one or more nucleic acids encoding a ZFN.
  • OTX2 polypeptide comprises an amino acid sequence having at least about 80% amino acid identity to the amino acid sequence set forth in SEQ ID NO: 13.
  • the recombinant vector of 144 wherein the mammalian cell is selected from a human cell, a rodent cell, and a non-human primate cell.
  • a recombinant vector comprising:
  • an exogenous nucleic acid comprising a nucleotide sequence encoding an OTX2 polypeptide; an exogenous nucleic acid comprising a nucleotide sequence encoding a PAX6 polypeptide;
  • an exogenous nucleic acid comprising a nucleotide sequence encoding an MITF polypeptide, wherein the vector is targeted to an AAVS1 locus, or ortholog thereof, or a CLYBL locus, or ortholog thereof, of a mammalian cell.
  • the recombinant vector of 149 wherein the vector does not comprise a nucleotide sequence encoding a transcription factor other than an OTX2 polypeptide, a PAX6 polypeptide, and/or an MITF polypeptide.
  • the recombinant vector of 152 wherein the recombinant vector is a donor vector for a gene editing system.
  • the recombinant vector of 153 wherein the gene editing system comprises a gene editing tool selected from a TALEN, a ZFN, one or more nucleic acids encoding a TALEN, and one or more nucleic acids encoding a ZFN.
  • OTX2 polypeptide comprises an amino acid sequence having at least about 80% amino acid identity to the amino acid sequence set forth in SEQ ID NO: 13.
  • the recombinant vector of any one of 149-158 wherein the nucleotide sequence encoding an OTX2 polypeptide is separated from the nucleotide sequence encoding a PAX6 polypeptide by a first ribosomal skipping sequence, and the nucleotide sequence encoding a PAX6 polypeptide is separated from the nucleotide sequence encoding a MITF polypeptide by a second ribosomal skipping sequence.
  • the recombinant vector of 159 wherein the first and/or second ribosomal skipping sequence encodes a 2A peptide selected from T2A, P2A, E2A, and F2A.
  • An isolated host cell comprising the recombinant vector of any one of 119- 160.
  • An isolated genetically modified host cell comprising an exogenous OTX2 nucleic acid, an exogenous PAX6 nucleic acid, and an exogenous MITF nucleic acid integrated into the genome of the host cell.
  • the host cell of 165 wherein the gene editing tool selected from a TALEN, a
  • ZFN ZFN
  • nucleic acids encoding a TALEN one or more nucleic acids encoding a ZFN.
  • a method of administering a retinal pigment epithelium comprising transplanting into a subject in need thereof a RPE generated by the method of any one of 1-106 or administering a therapeutically effective amount of a composition of 108 to a subject in need thereof.
  • RPE retinal pigment epithelium
  • transplanting comprises subretinal transplantation.
  • a method of treating a disease characterized by degeneration of retinal pigment epithelium comprising transplanting into a subject in need thereof a RPE generated by the method of any one of 1-106 or administering a therapeutically effective amount of a composition of 108 to a subject in need thereof.
  • AMD age-related macular degeneration
  • a macular dystrophy e.g., Stargardfs or Stargardt's-like disease
  • Best disease vitelliform macular dystrophy
  • adult vitelliform dystrophy or subtypes of retinitis pigmentosa e.g., vitelliform macular dystrophy or subtypes of retinitis pigmentosa.
  • Standard abbreviations may be used, e.g., bp, base pair(s); kb, kilobase(s); pl, picoliter(s); s or sec, second(s); min, minute(s); h or hr, hour(s); aa, amino acid(s); kb, kilobase(s); bp, base pair(s); nt, nucleotide(s); i.m, intramuscular(ly); i.p., intraperitoneal(ly); s.c., subcutaneous(ly); and the like.
  • Neural Induction (NI) medium comprised the following:
  • RPE maintenance medium comprised the following:
  • the RPE differentiation protocol using the AAVS hOPM cell line comprised the following:
  • iPSC culture conditions were maintained until start of the differentiation protocol.
  • OTX1, OTX2, MITF, PAX2, and PAX6 were expressed in separate viruses that were co-transfected in human iPSCs.
  • Plasmid pl372 was digested using Nhel and EcoRI. Using infusion cloning, each of the transcription factors attached to a T2A-GFP was cloned into the vector. Resulting plasmids were sequence verified before use.
  • HEK293T cells were seeded in an appropriate format. Either 7.5e6 was plated for a l5cm dish or 2.6e6 for a lOcm dish.
  • HEK cell media comprised of DMEM, high glucose, no glutamine 11960069; 10% FBS; l% NEAA; l% glutamax.
  • a 1.5 mL tube (Tube 1) was prepared by adding: a. l300 ⁇ L OptiMEM and 48 ⁇ L Mirus Reagent for a l5cm dish. b. 470 ⁇ L of OptiMEM and l8 ⁇ L Mirus Reagent for a lOcm dish. Incubate at RT for 5 minutes.
  • Tube 1 and Tube 2 were mixed together. The mix was left at room
  • Viral soup was placed in either a 15 ml or 50 ml tube.
  • the soup was filtered through a Milipore Millex-HP 0.45 ⁇ filter (hydrophobic PES) by adding the soup to a syringe with the filter attached.
  • the virus was aliquoted out into cryotubes for storage.
  • Viral supernatant was stored at -80°C until use.
  • CLYBL addgene ID 62196, 62197 Donor Vector Construction
  • cDNAs for each of the transcription factors human OTX1, human PAX6, and human MITF were cloned into a single poly-cistronic vector under the control of a Tet-0 -inducible promoter, with the coding sequence for each transcription factor separated by a T2A (ribosomal skipping sequence) (Tet-0 promoter driving OTX2- T2A-PAX6-T2A-MITF-STOP*) to provide for co-expression of human OTX1, human PAX6, and human MITF polypeptides.
  • T2A ribosomal skipping sequence
  • Two different vectors were prepared, one targeted to the AAVS1 genomic locus and the other targeted to the CLYBL genomic locus.
  • the vector map for the AAVS 1 -targeted vector is provided in FIG. 1.
  • PAX6 PMQNSHSGVNQLGGVFVNGRPLPDSTRQKIVELAHSGARPCDI
  • Positive selection marker comprised EF-la mCherry; gene trapping with puromycin selection (puromycin resistance only if properly integrated into target locus).
  • iPSCs were maintained so that they were actively dividing. One million well- singularized cells were collected in a pellet. Nucleofection was performed using Lonza 4D nucleofector using P3 solution and supplemented, pulse code EN138.
  • Reaction volume comprised as follows:
  • the protocol was adapted from: Zhu Y., Carido M., Meinhardt A., Kurth T., Karl M.O., Ader M., Tanaka E.M. (2013), Three-Dimensional Neuroepithelial Culture from Human Embryonic Stem Cells and Its Use for Quantitative Conversion to Retinal Pigment Epithelium, PLoS ONE, 8(1), art. no. e54552, for comparison with the methods of the present disclosure.
  • the protocol required mixing iPSCs into Matrigel suspension, followed by manual dissection of pigmented clumps pigmented cells.
  • One well of iPSC at approximately 80% confluency from a 6-well plate was used.
  • a 300pl aliquot of pure GFR-Matrigel was thawed out on ice.
  • the remaining cell suspension was repeated so that a number of discrete domes (up to 4) was formed per well of a 6 well plate.
  • the plate was incubated at 37°C for 30min. 2ml of Neural Differentiation medium was added with lx ROCKi per well.
  • Dav 5 The media was removed from the wells, washed with PBS and 4ml lx PBS/ well was added. The edge of a cell scraper was used to cut each dome into 4 pieces. Using a pipette, the pieces of Matrigel and PBS were transferred into a l5ml falcon tube. The Matrigel was allowed to settle at the bottom of the tube before the PBS was carefully removed. 5ml of Coming Cell Recovery Solution was added to the tube and the tube was incubated at 4°C for 40min to release the Neuroepithelial cysts from the Matrigel. The spheres were centrifuged at 400rpm for lmin and the cell recovery solution was carefully removed.
  • Neural Induction Media comprised the following: 195.5ml of Neurobasal medium, 195.5ml of DMEM, 4ml B27 supplement, 2ml N2 supplement, 400 ⁇ ⁇ - ME, and 2.5ml glutamax.
  • Protocol was adapted from Buchholz et al. Stem Cells Transl Med. 2013
  • RDM Retinal Differentiation Media
  • DMEM/F12 with Glutamax supplement; Invitrogen #10565018); IX N2 Supplement (#17502048, Invitrogen); IX B27 Supplement; and IX NEAA.
  • Nicotinamide (Sigma#N0636-l00G) had a working concentration of 10 mM.
  • bFGF ThermoFisher #13256029 lO ⁇ L was reconstituted to lO ⁇ L/mL and had a working concentration of 5ng/ml.
  • Vasoactive intestinal peptide (VIP) described in the original Buchholz et al. paper was omitted.
  • the substrate comprised of Matrigel, non-growth factor reduced (GF), Phenol Red-Free, (Coming, product #356237,10 mL).
  • the stock was made in -150-200 ⁇ L aliquots of concentrated Matrigel and stored at -20°C. For the working concentration, 1 aliquot was diluted into 12-12.5 mL serum-free media (just DMEM/F12). 0.5 mL/well was plated in a l2-well plate, and was aspirated right before seeding.
  • Matrigel w/ Phenol Red (Coming). The protein concentration varied from lot-to-lot, so bigger or smaller aliquots were created based on which lot.
  • Matrigel was thawed on ice at 4°C overnight. From 9 mg/mL stock, 200 ⁇ L aliquots were made. To make the working concentration, the 200 ⁇ L aliquot was added to l2ml DMEM/F12 to give 150 ⁇ L/mL final concentration.
  • Day 0 Day of stem cell passage for differentiation.
  • the Matrigel aliquot was mixed to homogeneity and diluted into 12-12.5mL DMEM/F12. 0.5mL/well was plated in a l2-well plate and the plate was incubated at room temperature for one hour. Any excess was aspirated off right before seeding cells.
  • RDM media was made by adding NOGGIN (50 ng/ml), DKK1 (10
  • hiPS cells were passaged 1:4 (into l2-well) depending on confluence. For every 6 well of cells to passage, 4 x l2-wells were plated for
  • Change media was changed to RDM media + NOGGIN (50 ng/ml); DKK1 (10 ng/ml); IGF1 (10 ng/ml); nicotinamide (lOmM) + bFGF (5 ng/mL).
  • the cells were re-plated on GFR-Matrigel-coated plates in RPE medium containing Y27632 at 1 x 10 ⁇ 5 cells/cm 2 .
  • Figures 17-22 depict vector maps of the plasmids used: Figure 17 depicts the CLYBL TALEN L plasmid and sequence (SEQ ID NO:30); Figure 18 depicts the CLYBL TALEN R plasmid and sequence (SEQ ID NO:3l); Figure 19 depicts the CLYBL hOPM plasmid and sequence (SEQ ID NO:32); Figure 20 depicts the AAVS1 TALEN L plasmid and sequence (SEQ ID NO:33); Figure 21 depicts the AAVS1 TALEN R plasmid and sequence (SEQ ID NO:34); and Figure 22 depicts the AAVS hOPM plasmid and sequence (SEQ ID NO:35).
  • Figure 23 depicts a schematic of the hOPM construct.
  • hOPM was inserted into a safe harbor locus (either CLYBL or AAVS1).
  • a safe harbor locus either CLYBL or AAVS1.
  • doxy cy cline was added, hOPM was transcribed. These transcription factors stayed on in mature RPE without doxy cy cline induction. This method required only media changes and yielded entire monolayers of RPE as opposed to small colonies that needed to be dissected.
  • Wild type and patient derived induced pluripotent stem cells were maintained in StemFit Basic02 (Ajinomoto, AS-BolR) with 5.8nM bFGF (Peprotech, 100-18B) in 6 well plates until the cells reached 70 percent confluency. Cells were then dissociated with Accutase (StemCell Technologies, 07920) and counted. A nucleofection solution consisting of l ⁇ L of the left and right TALENs as well as 2 ⁇ L of the hOPM plasmid and P3 supplement was prepared according to the instructions of the P3 Primary Cell 4D-Nucleofector X Kit L (Lonza, V4XP-3024).
  • Table 1 Primer list for genotyping clones for the hOPM construct
  • Induced pluripotent stem cells were dissociated with Accutase (StemCell Technologies, 07920) and passaged into a 96-well plate coated with Matrigel (Coming, 356231). Cells were seeded at a density of roughly 48,000 cells per cm 2 and maintained in StemFit Basic02 (Ajinomoto, AS-BolR) with lOuM ROCK inhibitor (Selleckchem, S1049) for 24 hours. The media was then changed to StemFit Basic02 (Ajinomoto, AS-BolR) and the cells were allowed to grow to 90 to 100 percent confluency.
  • NI Neural Induction
  • RPEMM RPE Maintenance Media
  • Dox Doxy cy cline
  • LSB LDN-193189 + SB-431542
  • Induction Media comprised of 49% DMEM F12 (ThermoFisher Scientific).
  • RPE Maintenance Media comprised of Knockout DMEM (Life Technologies, 10829-018), 20% Knockout Serum Replacement (ThermoFisher Scientific, 10828028), 1% Glutamax (Life Technologies, 35050-061), l% NEAA (Life Technologies, 11140-050), 0.18% 55mM B-mercaptoethanol (Life Technologies, 10829-018), 20% Knockout Serum Replacement (ThermoFisher Scientific, 10828028), 1% Glutamax (Life Technologies, 35050-061), l% NEAA (Life Technologies, 11140-050), 0.18% 55mM B-mercaptoethanol (Life
  • Pig RPE Maintenance Media comprised of DMEM (Coming, 10-013-CV), 1% NEAA (Life Technologies, 11140- 050), 1% FBS (ATCC, 30-2020), and 0.4% of THT supplement, which was prepared using 99ml Earle’s Balanced Salt Solution (Life Technologies, 14155-063), l2.5g taurine (Sigma Aldrich, 107-35-7), lmg hydrocortisone (Sigma Aldrich, 50237), 0.65 ⁇ L liothyronine (Sigma Aldrich, 1368008).
  • the cells were fed with this media every Monday and Thursday until Day 60 when the cells were taken off doxy cy cline induction. Cells past Day 60 continued to be maintained with Pig RPE Maintenance Media with biweekly feedings. The best wells from this 96 well plate optimization were then expanded into a whole plate format to validate the optimization protocol.
  • WTB6.13 AAVS hOPM C4 cell pellets were collected at different points in the differentiation starting with day zero as induced pluripotent stem cells and including iPSC-RPE cells at week 6, week 8, and week 12 of the differentiation.
  • Droplets were generated by the Bio-Rad QX100 Droplet Generator (Bio-Rad, 186-3001) from 20 ⁇ L reaction mixtures and 70 ⁇ L Droplet Generator Oil (Bio-Rad, 186-3005) to result in about 35 ⁇ L of product.
  • the PCR plate was sealed using the PX1 PCR Plate Sealer (Bio-Rad, 181-4000), a C1000 Touch Thermal Cycler (Bio- Rad, 185-1196) was used for the ddPCR reaction, and the QX100 Droplet Reader (Bio-Rad, 186-4003) for the analysis of the droplets.
  • the threshold was set on Quantasoft Software (Bio-Rad, 186-4011) and the data were normalized to the housekeeping gene, GAPDH, and graphed on excel with respect to the iPSC concentration.
  • OTX1, OTX2, MITF, PAX2, and PAX6 were initially tested using individual lentiviral vectors. Five weeks of transcription factor overexpression in human iPSCs resulted in the generation of RPE as shown in Figure 2. Based on these preliminary positive results an attempt was made to co-express transcription factors in a single vector to provide more uniform expression. It was tested whether OTX1 and PAX2 could be eliminated, while maintaining RPE generation, since they have redundant roles with the remaining three factors.
  • AAVS1 allowing uniform expression in iPSCs and differentiation of iPSCs to a monolayer RPE. See, e.g., Figures 4-8.
  • This vector is referred to herein as hOPM- AAVS.
  • hOPM-CLYBL CLYBL locus
  • the CLYBL locus has been shown to have higher levels of expression with other transgenes.
  • expression may be increased by combining multiple integration sites.
  • by combining higher transcription factor dosing, with different doxy cy cline dosing the optimal timing and extent of transcription factor over-expression can be determined.
  • hOPM-AAVS or hOPM-CLYBL An advantage of this system is that the Tet system keeps the transcription factors (OTX2, PAX6 and MITF) off in the pluripotent state, allowing the cell line to be propagated prior to differentiation. This allows for unlimited production of source material without the need for repeated introduction of transcription factors via viral delivery.
  • An additional advantage of this system is that the timing of OPM expression can be controlled.
  • the timing of OPM expression can be controlled.
  • the cells peeled off the plate and died off (data not shown).
  • This system allows one to precisely control the timing of induction to provide for an optimized system.
  • the inducible system also allows for the expression of OPM to be turned off after RPE is formed, allowing the RPE to be studied without artificial expression of OPM.
  • BEST1 cDNA was detected at the exon 6/7 junction at 6 weeks, 8 weeks, 10 weeks, and 12 weeks of differentiation using the hOPM-AAVS vector in the presence of doxy cy cline induction as described herein. In contrast, in the absence of doxy cy cline induction, but otherwise using the same conditions, BEST1 cDNA was not detected until 10 weeks using the hOPM-AAVS vector. No BEST1 cDNA was detected at 8 weeks at the exon 6/7 junction in the absence of vector, either in the presence or absence of doxy cy cline.
  • the OPM-RPE is significantly easier to make (requires fewer media changes, and there is no need to re-plate cells) and does not require the expensive recombinant hormones used in the chemical induction methods.
  • the OPM-RPE is more uniform than that provided by other available methods (even methods taking twice as long) which form RPE in patches, that are at varying degrees of maturity. See, e.g., Figure 10, which provides a comparison showing pigmented colonies at day 59 of differentiation using the protocol described in Figure 10 (top) relative to a pigmented monolayer of RPE cells produced at day 56 of the transcription factor induction protocol described herein using the hOPM-AAVS vector (bottom).
  • Figure 24 depicts a comparison of the Leach et al. and Zhu et al. protocols described herein with a method of the present disclosure, which required only media changes and yielded entire monolayers of RPE as opposed to small colonies that needed to be dissected.
  • iPSCs were seeded, grown to 100% confluency in 96 well plate format, and conditioned for optimization with Dual Smad inhibition (Figure 26). RPE cells were grown to maturity (around 60 days) and best wells were picked to expand.
  • RPE are derived from the neurectodermal lineage.
  • LSB, (LDN + SB) are small molecule dual-smad inhibitors used to specify the neurectodermal state and are more directed than use of NI.
  • Some of the cell lines preferred the“Neural Induction” medium from Days 0-6 while others preferred“RPE Maintenance Media.”
  • the timing of Doxycycline addition also varied between cell lines with some lines preferring Doxycycline induction beginning on Day 0, while others preferred it on Day 2 or Day 4.
  • the concentration of LSB that these cell lines preferred varied with one cell line differentiating well without LSB and others differentiating well with O. lx and 0.05x concentrations of LSB. Timing and duration of LSB also varied. Most lines with LSB in their differentiation protocol preferred it at Day 0 yet the preferred length of LSB treatment varied. Some cell lines performed well with LSB treatment until Day 6 and other cell lines performed better with LSB treatment terminated on Day 2 or Day 4.
  • Figure 29 depicts images of RPE development at 60+ days for WTB and WTC cell lines, showing the dark pigmentation and hexagonal or cobblestone morphology that are characteristic of RPE.
  • Figure 30 depicts images of RPE development at 60+ days for BTP6 and BTP7 cell lines, showing the dark pigmentation and hexagonal or cobblestone morphology that are characteristic of RPE.
  • Figure 31 depicts images of RPE development at 60+ days for BTP8 and BTP10 cell lines, showing the dark pigmentation and hexagonal or cobblestone morphology that are characteristic of RPE.
  • Figure 32 depicts images showing the stage of RPE development that are stained with generic tight junction proteins occludin and phalloidin. RPE formed a monolayer with strong tight junctions between cells.
  • Figures 33-35 depict images showing mature induced pluripotent stem cell
  • (iPS)-derived RPE The image showing RPE development at day 60 for WTB Traffic Light was taken at 5x magnification ( Figure 33, left). The monolayer of RPE was shown forming a fluid-filled dome, indicating that the RPE was carrying out the function of transporting fluid. The image showing RPE development at day 60+ for BTP8 was taken at 20x magnification and shows the characteristic hexagonal or cobblestone shape and dark pigmentation of RPE ( Figure 33, right).
  • Figure 34 depicts images showing RPE development at -day 45 for BTP10 (left) and BTP6 (right). These images were taken at lOx magnification and show RPE characteristic hexagonal shape as well as pigmentation beginning to develop. Although some cells did not have pigmentation yet, they had the correct hexagonal morphology and would begin to develop pigment in the coming weeks.
  • Figure 35 depicts images showing RPE development at day -60 for WTB (left) and WTC (right). This image was taken at 40x magnification and shows the dark pigmentation and hexagonal shape of these RPE.
  • Figure 36 depicts gene expression results using ddPCR for RPE specific genes.
  • the probes used included SERPINF, BEST1, MITF RLBP1, and RPE65.
  • SERPINF refers to“PEDF” or“Pigment Epithelium Derived Factor,” and is secreted apically from RPE cells, antiangiogenic.
  • BEST1 encodes bestrophinl which is a calcium activated chloride channel localized to the basolateral membrane of RPE.
  • MITF encodes a transcription factor that is responsible for turning on RPE specific genes and pigmentation genes.
  • RLBP1 encodes a protein that is a ligand to 1 l-cis retinal; a component of the visual cycle.
  • RPE65 is also a component of the visual cycle, and is an isomerohydrolase that regenerates visual pigment.
  • RPE specific gene expression data in WTB AAVS hOPM C4 is shown as a function of timepoint mRNA/iPSC over age.
  • the RPE specific genes depict exponential fold increase from iPSCs.

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Abstract

La présente invention concerne des procédés de génération d'épithélium pigmentaire rétinien (EPR) à partir de cellules souches pluripotentes induites (iPSC). La présente invention concerne également un EPR généré à l'aide d'un procédé selon l'invention ainsi que des procédés d'utilisation d'EPR généré selon les procédés décrits. La présente invention concerne également des vecteurs recombinants utiles pour générer un EPR selon les procédés décrits.
PCT/US2018/061100 2017-11-14 2018-11-14 Procédés de génération d'épithélium pigmentaire rétinien (epr) WO2019099552A1 (fr)

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WO2020209800A3 (fr) * 2019-04-11 2020-11-12 Agency For Science, Technology And Research Procédé de modification d'un état de différenciation d'une cellule
ES2823398A1 (es) * 2019-11-05 2021-05-06 Consejo Superior Investigacion Composición biopolimérica, procedimiento para su preparación y uso de la misma
CN113512564A (zh) * 2021-08-05 2021-10-19 呈诺再生医学科技(珠海横琴新区)有限公司 以iPSC来源的细胞为载体、基因治疗湿性年龄相关性黄斑变性
WO2023091708A1 (fr) * 2021-11-18 2023-05-25 The Brigham And Women's Hospital, Inc. Modèles de protéinopathie induite
CN117384846A (zh) * 2023-12-08 2024-01-12 南京艾尔普再生医学科技有限公司 一种多能干细胞来源的rpe细胞、制剂、药物组合物、试剂、方法及试剂盒
EP4110899A4 (fr) * 2020-02-28 2024-03-20 Riken Procédé de préparation d'une cellule épithéliale pigmentaire rétinienne

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2020209800A3 (fr) * 2019-04-11 2020-11-12 Agency For Science, Technology And Research Procédé de modification d'un état de différenciation d'une cellule
ES2823398A1 (es) * 2019-11-05 2021-05-06 Consejo Superior Investigacion Composición biopolimérica, procedimiento para su preparación y uso de la misma
EP4110899A4 (fr) * 2020-02-28 2024-03-20 Riken Procédé de préparation d'une cellule épithéliale pigmentaire rétinienne
CN113512564A (zh) * 2021-08-05 2021-10-19 呈诺再生医学科技(珠海横琴新区)有限公司 以iPSC来源的细胞为载体、基因治疗湿性年龄相关性黄斑变性
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WO2023091708A1 (fr) * 2021-11-18 2023-05-25 The Brigham And Women's Hospital, Inc. Modèles de protéinopathie induite
CN117384846A (zh) * 2023-12-08 2024-01-12 南京艾尔普再生医学科技有限公司 一种多能干细胞来源的rpe细胞、制剂、药物组合物、试剂、方法及试剂盒
CN117384846B (zh) * 2023-12-08 2024-04-26 南京艾尔普再生医学科技有限公司 一种多能干细胞来源的rpe细胞、制剂、药物组合物、试剂、方法及试剂盒

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