Compositions and methods for enriching photoreceptors

Abstract

Provided herein are methods of enriching for photoreceptors and photoreceptor precursor cells from mixed cell populations. Such enriched populations of photoreceptors and photoreceptor precursor cells can be used as cell therapies for the treatment of retinal disorders.

Description

COMPOSITIONS AND METHODS FOR ENRICHING PHOTORECEPTORSCROSS-REFERENCE

[0001] This application claims the benefit of and priority to U.S. Provisional Patent Application No. 63 / 718,960, filed November 11, 2024, which is incorporated by reference in its entirety herein.FIELD OF THE INVENTION

[0001] The invention relates to the field of identification of photoreceptors (e.g., cone photoreceptors) in populations of cells. The invention also relates to the field of cell-based therapies for treating ocular disorders.BACKGROUND

[0002] Ocular disorders, such as inherited retinal diseases (IRDs), age-related macular degeneration (AMD), Stargardt disease, and geographic atrophy (GA), may lead to partial or complete vision loss and are characterized by progressive and irreversible loss of photoreceptors. In the advanced stages of these diseases, gene replacement therapies are not effective because the number of target cells is insufficient. In this context, the feasibility of cell replacement therapy has already been demonstrated in animal models and constitutes a viable alternative for restoring vision.

[0003] While primary retina transplantation has been posed as a potential therapy in these cases, it is limited by the availability of donor tissues. Stem cells, including human induced pluripotent stem cells (hiPSCs), constitute an expandable and renewable cell source from which photoreceptors can be produced. Yet, the production of bona fide functional photoreceptor cells with high yields remains a challenge. Moreover, although certain 2D differentiation strategies have been shown to generate high yields of photoreceptors (>90%), those strategies produce primarily cells with molecular profiles akin to those of rod photoreceptors.

[0004] Another approach to generate photoreceptors is the use of retinal organoid technology. Several methods exist for the generation of self-organizing 3D retinal organoids. Produced by recapitulating retinal development as it occurs in vivo, stem cell-derived human retinal organoids (HRO) yield cells that exhibit molecular profiles with a high degree of similarity to those of the cells found in primary tissues, including ganglion, amacrine, bipolar, horizontal, Muller, and cone and rod photoreceptor cells. Retinal organoids, like primary retinal tissues, can be transplanted. However, clinical implementation is challenging due to the surgicalprocedure being highly invasive and requiring a trained ophthalmic surgeon. Additionally, the generation, identification, and isolation of HRO and regions thereof suitable for transplantation, which must both exhibit a well-defined morphology, also requires advanced skills.

[0005] An alternative approach involves the dissociation of HRO into a single-cell suspension. However, it remains unclear whether the presence of non-photoreceptor cells in such suspensions may hinder the survival of a graft, its integration into a host tissue, and / or its subsequent contribution to visual recovery. Hence, the need to isolate photoreceptor precursors from an HRO constitutes a potential challenge for this approach to be successful.

[0006] Thus, there is a need in the art for methods to isolate photoreceptors (e.g., cone photoreceptors) from populations of cells containing more than one cell type, as is the case for stem-cell derived HRO. Further, there is a need in the art for improved photoreceptor transplantation strategies for ocular disorders such as IRDs, AMD, Stargardt disease, and GA.SUMMARY OF THE INVENTION

[0007] The disclosure relates to the discovery of novel photoreceptor-specific and cone photoreceptor-specific cell surface markers for the enrichment of photoreceptors (e.g., cone photoreceptors) and precursors thereof from mixed populations of cells. Accordingly, in certain aspects, the disclosure relates to methods of enriching photoreceptor cells and photoreceptor precursor cells from a mixed population of cells. Furthermore, the disclosure relates to a method of treating a retinal disorder, the method comprising administering the population of enriched photoreceptor cells.

[0008] In one aspect, the disclosure relates to a method of enriching photoreceptor cells and / or photoreceptor precursor cells from a mixed population of cells, the method comprising: contacting the mixed population of cells with a JAM-B binding agent, allowing the photoreceptor cells and / or photoreceptor precursor cells to be bound by the JAM-B binding agent, and separating at least a portion of the bound photoreceptor cells and / or photoreceptor precursor cells from the mixed population of cells, thereby obtaining a population of cells enriched for photoreceptor cells and / or photoreceptor precursor cells.

[0009] In certain embodiments, the method further comprises performing a second enrichment by: contacting the enriched photoreceptor cells and / or photoreceptor precursor cells with a CD26 and / or CD133 binding agent, allowing the photoreceptor cells and / or photoreceptor precursor cells to be bound by a CD26 and / or CD133 binding agent, respectively, and separating at least a portion of the bound photoreceptor cells and / or photoreceptor precursor cells from theenriched photoreceptor cells and / or photoreceptor precursor cells to obtain a population of cells further enriched for photoreceptor cells and / or photoreceptor precursor cells.

[0010] In certain embodiments, the JAM-B binding agent is an antibody or a nanobody.

[0011] In certain embodiments, the CD26 and / or CD133 binding agent is an antibody or a nanobody.

[0012] In certain embodiments, the method enriches for photoreceptor cells. In certain embodiments, the photoreceptor cells are cone photoreceptor cells.

[0013] In certain embodiments, the method enriches for photoreceptor precursor cells. In certain embodiments, the photoreceptor precursor cells are cone photoreceptor precursor cells.

[0014] In certain embodiments, the mixed population of cells comprises differentiated human induced pluripotent stem (iPS) or embryonic stem (ES) cells.

[0015] In certain embodiments, the mixed population of cells comprises human retinal organoid cells.

[0016] In certain embodiments, the separating step is performed using fluorescence-activated cell sorting (FACS), magnetic-activated cell sorting (MACS), and / or cell panning.

[0017] In certain embodiments, the enriched population of cells comprises at least about 80%, at least about 85%, at least about 90%, or at least about 95% photoreceptor and / or photoreceptor precursor cells.

[0018] In another aspect, the disclosure relates to a method of enriching photoreceptor cells and / or photoreceptor precursor cells in a mixed population of cells, the method comprising: contacting the mixed population of cells with a CD26 binding agent and, optionally, a JAM-B binding agent, allowing the photoreceptor cells and / or photoreceptor precursor cells to be bound by the CD26 binding agent and the JAM-B binding agents, if present, and separating at least a portion of the bound photoreceptor cells and / or photoreceptor precursor cells from the mixed population of cells, thereby obtaining a population of cells enriched for photoreceptor cells and / or photoreceptor precursor cells, wherein the mixed population is enriched using only the CD26 binding agent and, optionally the JAM-B binding agent, and wherein the method does not include contacting the mixed population of cells or the enriched population of cells with a CD29 binding agent or a CD15-SSEA1 binding agent.

[0019] In certain embodiments, the CD26 binding agent and / or the JAM-B binding agent is an antibody or a nanobody.

[0020] In certain embodiments, the method enriches for photoreceptor cells. In certain embodiments, the photoreceptor cells are cone photoreceptor cells.

[0021] In certain embodiments, the method enriches for photoreceptor precursor cells. In certain embodiments, the photoreceptor precursor cells are cone photoreceptor precursor cells.

[0022] In certain embodiments, the mixed population of cells comprises differentiated human iPS or ES cells.

[0023] In certain embodiments, the mixed population of cells comprises human retinal organoid cells.

[0024] In certain embodiments, the separating step is performed using FACS, MACS, and / or cell panning.

[0025] In certain embodiments, the enriched population of cells comprises at least about 80%, at least about 85%, at least about 90%, or at least about 95% photoreceptor and / or photoreceptor precursor cells.

[0026] In another aspect, the disclosure relates to a method of enriching photoreceptor cells and / or photoreceptor precursor cells in a mixed population of cells, the method comprising: contacting the mixed population of cells with a CD133 binding agent and, optionally, a JAM-B binding agent, allowing the photoreceptor cells and / or photoreceptor precursor cells to be bound by the CD 133 and JAM-B binding agents, if present, and separating at least a portion of the bound photoreceptor cells and / or photoreceptor precursor cells from the mixed population of cells, thereby obtaining a population of cells enriched for photoreceptor cells and / or photoreceptor precursor cells, wherein the mixed population is enriched using only the CD133 binding agent and optionally the JAM-B binding agent, and wherein the method does not include contacting the mixed population of cells or the enriched population of cells with a CD29 binding agent or a CD15-SSEA1 binding agent.

[0027] In certain embodiments, the CD133 binding agent and / or the JAM-B binding agent is an antibody or a nanobody.

[0028] In certain embodiments, the method enriches for photoreceptor cells. In certain embodiments, the photoreceptor cells are cone photoreceptor cells.

[0029] In certain embodiments, the method enriches for photoreceptor precursor cells. In certain embodiments, the photoreceptor precursor cells are cone photoreceptor precursor cells.

[0030] In certain embodiments, the mixed population of cells comprises differentiated human iPS or ES cells.

[0031] In certain embodiments, the mixed population of cells comprises human retinal organoid cells.

[0032] In certain embodiments, the separating step is performed using FACS, MACS, and / or cell panning.

[0033] In certain embodiments, the enriched population of cells comprises at least about 80%, at least about 85%, at least about 90%, or at least about 95% photoreceptor and / or photoreceptor precursor cells.

[0034] In another aspect, the disclosure relates to a population of cells obtained from differentiated iPS or ES cells or retinal organoid cells wherein at least 80%, at least 85%, at least 90%, or at least 95% of the population of cells are photoreceptor and / or photoreceptor precursor cells.

[0035] In certain embodiments, the photoreceptor and / or photoreceptor precursor cells express JAM-B on their surface.

[0036] In certain embodiments, the population of cells was obtained using the method of any one of the foregoing aspects or embodiments.

[0037] In another aspect, the disclosure relates to a method of treating a retinal disorder, the method comprising, administering the population of cells of any one of the foregoing aspects or embodiments.

[0038] In certain embodiments, the method comprises administering transvitreally or via a suprachoroidal route.BRIEF DESCRIPTION OF THE DRAWINGS

[0039] These and other features, aspects, and advantages of the present invention will become better understood with regard to the following description, and accompanying drawings, where:

[0040] FIG. 1A is a set of scatterplots showing the fraction of cells positive for mCherry (x- axis) and CD26-fluorescein isothiocyanate (FITC) (fluorophore; y-axis) in cone-rod homeobox (Crx)-mCherry induced pluripotent stem (iPS) cell-derived retinal organoids after dissociation into single cell suspensions as measured using fluorescence-activated cell sorting (FACS). The three conditions assessed included dissociated iPS cell-derived retinal organoid cells without labelling with the CD26 antibody (“Unstained”), dissociated iPS cell-derived retinal organoid cells labelled with the CD26 antibody (“Pre-sort”), and dissociated iPS cell-derived retinal organoid cells labelled with the CD26 antibody after enrichment with a MACSQuant Tyto instrument (“Enriched”). FIG. IB is a bar plot depicting the number of CD26- FITC-positive cells in three independent differentiations of the Crx-mCherry iPS cell line towards retinal organoids. P-values were calculated using a Student’s t-test with Welch’s correction between the ‘Pre-sort’ and ‘Enriched’ conditions as described in FIG. 1 A. FIG. 1C is a bar plot depicting the number of CD26-FITC and mCherry double-positive cells in three independent retinal organoiddifferentiations from the Crx-mCherry iPS cell line. P-values were calculated using a Student’s t- test with Welch’s correction between the ‘Pre-sort’ and ‘Enriched’ conditions as described in FIG 1A FIG. ID is a set of scatterplots showing the fraction of cells positive for mCherry (x- axis) and CD26-FITC (y-axis) in cone arrestin (Car)-mCherry iPS cell-derived retinal organoids after dissociation into a single cell suspension as measured using FACS. The three conditions assessed included dissociated iPS cell-derived retinal organoid cells without labelling with the CD26 antibody (“Unstained”), dissociated iPS cell-derived retinal organoid cells labelled with the CD26 antibody (“Pre-sort”), and dissociated iPS cell-derived retinal organoid cells labelled with the CD26 antibody after enrichment with a MACSQuant Tyto instrument (“Enriched”). FIG. IE is a bar plot depicting the number of CD26-FITC-positive cells in three independent differentiation runs of the Car-mCherry iPS cell line towards retinal organoids. P-values were calculated using a Student’s t-test with Welch’s correction between the ‘Pre-sort’ and ‘Enriched’ conditions as described in FIG. ID. FIG. IF is a bar plot depicting the number of CD26-FITC and mCherry double-positive cells in three independent differentiation runs of the Car-mCherry iPS cell line towards retinal organoids. P-values were calculated using a Student’s t-test with Welch’s correction between the ‘Pre-sort’ and ‘Enriched’ conditions as described in FIG. ID.

[0041] FIG. 2A is a set of scatterplots showing the fraction of cells positive for mCherry (x- axis) and CD133-PE-Vio770 (fluorophore; y-axis) in Crx-mCherry induced pluripotent stem (iPS) cell-derived retinal organoids as measured after dissociation into single cell suspensions using FACS. The three conditions assessed included dissociated iPS cell-derived retinal organoid cells without labelling with the CD133 antibody (“Unstained”), dissociated iPS cell-derived retinal organoid cells labelled with the CD133 antibody (“Pre-sort”), and dissociated iPS cell- derived retinal organoid cells labelled with the CD133 antibody after enrichment with a MACSQuant Tyto instrument (“Enriched”). FIG. 2B is a bar plot depicting the number of CD133- PE-Vio770-positive cells in three independent differentiation runs of the Crx-mCherry iPS cell line towards retinal organoids. P-values were calculated using a Student’s t-test with Welch’s correction between the ‘Pre-sort’ and ‘Enriched’ conditions as described in FIG. 2A. FIG. 2C is a bar plot depicting the number of CD133-PE-Vio770 and mCherry double-positive cells in three independent differentiation runs of the Crx-mCherry iPS cell line towards retinal organoids. P-values were calculated using a Student’s t-test with Welch’s correction between the ‘Pre-sort’ and ‘Enriched’ conditions as described in FIG. 2A. FIG. 2D is a set of scatterplots showing the fraction of cells positive for mCherry (x-axis) and CD133-PE-Vio770 (y-axis) in Car-mCherry iPS cell-derived retinal organoids after dissociation into a single cell suspension as measured using FACS. The three conditions assessed included dissociated iPS cell-derivedretinal organoid cells without labelling with the CD133 antibody (“Unstained”), dissociated iPS cell-derived retinal organoid cells labelled with the CD133 antibody (“Pre-sort”), and dissociated iPS cell-derived retinal organoid cells labelled with the CD133 antibody after enrichment with a MACSQuant Tyto instrument (“Enriched”). FIG. 2E is a bar plot depicting the number of CD133-PE-Vio770 -positive cells in three independent differentiation runs of the Car-mCherry iPS cell line towards retinal organoids. P-values were calculated using a Student’s t-test with Welch’s correction between the ‘Pre-sort’ and ‘Enriched’ conditions as described in FIG. 2D. FIG. 2F is a bar plot depicting the number of CD133-PE-Vio770 and mCherry double-positive cells in three independent differentiation runs of the Car-mCherry iPS cell line towards retinal organoids. P-values were calculated using a Student’s t-test with Welch’s correction between the ‘Pre-sort’ and ‘Enriched’ conditions as described in FIG. 2D.

[0042] FIG. 3A is a set of scatterplots showing the fraction of cells positive for mCherry (x- axis) and JAM-B-allophycocyanin (APC, fluorophore; y-axis) in Crx-mCherry iPS cell-derived retinal organoids as measured after dissociation into a single cell suspension using FACS. The three conditions assessed included dissociated iPS cell-derived retinal organoid cells without labelling with the JAM-B antibody (“Unstained”), dissociated iPS cell-derived retinal organoid cells labelled with the JAM-B antibody (“Pre-sort”), and dissociated iPS cell-derived retinal organoid cells labelled with the JAM-B antibody after enrichment with a MACSQuant Tyto instrument (“Enriched”). FIG. 3B is a bar plot depicting the number of JAM-B-APC-positive cells in three independent differentiation runs of the Crx-mCherry iPS cell line towards retinal organoids. P-values were calculated using a Student’s t-test with Welch’s correction between the ‘Pre-sort’ and ‘Enriched’ conditions as described in FIG. 3 A. FIG. 3C is a bar plot depicting the number of JAM-B-APC and mCherry double-positive cells in three independent differentiation runs of the Crx-mCherry iPS cell line towards retinal organoids. P-values were calculated using a Student’s t-test with Welch’s correction between the ‘Pre-sort’ and ‘Enriched’ conditions as described in FIG. 3A. FIG. 3D is a set of scatterplots showing the fraction of cells positive for mCherry (x-axis) and JAM-B-APC (y-axis) in Car-mCherry iPS cell-derived retinal organoids after dissociation into a single cell suspension as measured using FACS. The three conditions assessed included dissociated iPS cell-derived retinal organoid cells without labelling with the JAM-B antibody (“Unstained”), dissociated iPS cell-derived retinal organoid cells labelled with the JAM-B antibody (“Pre-sort”), and dissociated iPS cell -derived retinal organoid cells labelled with the JAM-B antibody after enrichment with a MACSQuant Tyto instrument (“Enriched”). FIG. 3E is a bar plot depicting the number of JAM-B-APC -positive cells in three independent differentiation runs of the Car-mCherry iPS cell line towards retinal organoids. P-values werecalculated using a Student’s t-test with Welch’s correction between the ‘Pre-sort’ and ‘Enriched’ conditions as described in FIG. 3D. FIG. 3F is a bar plot depicting the number of JAM-B-APC and mCherry double-positive cells in three independent differentiation runs of the Car-mCherry iPS cell line towards retinal organoids. P-values were calculated using a Student’s t-test with Welch’s correction between the ‘Pre-sort’ and ‘Enriched’ conditions as described in FIG. 3D.DETAILED DESCRIPTION

[0043] The disclosure relates to the discovery of novel photoreceptor-specific and cone photoreceptor-specific cell surface markers and improved methods for the enrichment of cone photoreceptors from populations of cells. Accordingly, in certain aspects, the disclosure relates to methods of enriching photoreceptor cells and / or photoreceptor precursor cells from a mixed population of cells, the method comprising: contacting the mixed population of cells with a JAM- B, CD26, and / or CD133 binding agent, respectively, allowing the photoreceptor cells and / or photoreceptor precursor cells to be bound by the respective binding agent, and separating at least a portion of the bound photoreceptor cells and / or photoreceptor precursor cells from the mixed population of cells, thereby obtaining a population of cells enriched for photoreceptor cells and / or photoreceptor precursor cells.

[0044] The disclosure also relates, in part, to cells, such as enriched photoreceptors and / or photoreceptor precursor cells for treating a retinal disorder. Accordingly, in certain aspects, the disclosure relates to a method of treating a retinal disorder, the method comprising administering the population of enriched photoreceptor cells.

[0045] The details of various embodiments of the invention are set forth in the description below. Other features, objects, and advantages of the invention will be apparent from the description and the drawings and from the claims.I. Definitions

[0046] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the art to which the claimed subject matter belongs. Generally, nomenclatures utilized in connection with, and techniques of, immunology, oncology, cell and tissue culture, molecular biology, and protein and oligonucleotide or polynucleotide chemistry and hybridization described herein are those well-known and commonly used in the art. It is to be understood that the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of anysubject matter claimed. The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0047] As used herein, singular forms “a,” “and,” and “the” include plural referents unless the context clearly indicates otherwise. Thus, for example, reference to “an embryonic stem cell” includes a plurality of embryonic stem cells and reference to “an embryonic stem cell” in some embodiments includes multiple embryonic stem cells, and so forth.

[0048] As used herein, all numerical values or numerical ranges include whole integers within or encompassing such ranges and fractions of the values or the integers within or encompassing ranges unless the context clearly indicates otherwise. Thus, for example, reference to a range of 90-100%, includes 91%, 92%, 93%, 94%, 95%, 96%, 97%, etc., as well as 91.1%, 91.2%, 91.3%, 91.4%, 91.5%, etc., 92.1%, 92.2%, 92.3%, 92.4%, 92.5%, etc., and so forth. In another example, reference to a range of 1-5,000 fold includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, fold, etc., as well as 1.1, 1.2, 1.3, 1.4, 1.5 fold, etc., 2.1, 2.2, 2.3, 2.4, 2.5 fold, etc., and so forth.

[0049] “About” a number, as used herein, refers to range including the number and ranging from 10% below that number to 10% above that number. “About” a range refers to 10% below the lower limit of the range, spanning to 10% above the upper limit of the range.

[0050] The terms “recipient,” “individual,” “subject,” “host,” and “patient,” are used interchangeably herein and refer to any mammalian subject for whom diagnosis, treatment, or therapy is desired, particularly humans. “Mammal” for purposes of treatment refers to any animal classified as a mammal, including humans, domestic and farm animals, and laboratory, zoo, sports, or pet animals, such as dogs, horses, cats, cows, sheep, goats, pigs, mice, rats, rabbits, guinea pigs, monkeys, etc. In some embodiments, the mammal is a human.

[0051] As used herein, the terms “treatment,” “treating,” and the like, in some cases, refer to administering an agent (e.g., a population of cells) or carrying out a procedure, for the purposes of obtaining an effect. In some embodiments, the effect is prophylactic in terms of completely or partially preventing a disease or symptom thereof and / or is therapeutic in terms of effecting a partial or complete cure for a disease and / or symptoms of the disease. “Treatment,” as used herein, includes treatment of a disease or disorder (e.g., a retinal disorder) in a mammal, particularly in a human, and includes: (a) preventing the disease or a symptom of a disease from occurring in a subject which is predisposed to the disease but has not yet been diagnosed as having it (e.g., including diseases that are associated with or caused by a primary disease); (b) inhibiting the disease, i.e., arresting its development; and (c) relieving the disease, i.e., causing regression of the disease. In some embodiments, treating refers to any indicia of success in thetreatment or amelioration or prevention of a retinal disorder, including any objective or subjective parameter such as abatement; remission; diminishing of symptoms or making the disease condition more tolerable to the patient; slowing in the rate of degeneration or decline; or making the final point of degeneration less debilitating. The treatment or amelioration of symptoms is based on one or more objective or subjective parameters, including the results of an examination by a physician. Accordingly, the term “treating” includes the administration of the compounds or agents of the present disclosure to prevent or delay, to alleviate, or to arrest or inhibit development of the symptoms or conditions associated with diseases (e.g., a retinal disorder). The term “therapeutic effect” refers to the reduction, elimination, or prevention of the disease, symptoms of the disease, or side effects of the disease in the subject. A subject is “treated” for a disease or disorder if, after receiving a therapeutic amount of a population of cells of the present disclosure, the patient shows observable and / or measurable change in a parameter or symptom of the disease or disorder.

[0052] In some embodiments, administration of a therapeutic agent in a prophylactic method occurs prior to the manifestation of symptoms of an undesired disease or disorder, such that the disease or disorder is prevented or, alternatively, delayed in its progression. Thus, when used in conjunction with prophylactic methods, the term “therapeutically effective” means that, after treatment, a smaller number of subjects (on average) develop the undesired disease or disorder or exhibit less severe symptoms or slower progress towards more severe symptoms.

[0053] The terms “embryonic stem cells” and “ES cells” refer to embryo-derived cells. More specifically they refer to cells isolated from the inner cell mass of blastocysts or morulae that have been serially passaged as cell lines. The term also 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, even when non-embryonic cells are used in the process.

[0054] The term “human embryonic stem cells” (hESCs) is used herein as it is used in the art. This term includes cells derived from the inner cell mass of human blastocysts or morulae that have been serially passaged as cell lines. The hESCs may, in some embodiments, be derived from fertilization of an egg cell with sperm or DNA, nuclear transfer, parthenogenesis, or by means to generate hESCs with homozygosity in the human leukocyte antigen (HLA) region. HESCs are also cells 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 a cell.

[0055] As used herein, the term “pluripotent stem cells” (PSC) includes embryonic stem (ES) cells, embryo-derived stem cells, and induced pluripotent stem cells (iPSC), regardless of the method by which the pluripotent stem cells are derived.

[0056] As used herein, the terms “induced pluripotent stem cells” and “iPSCs,” mean that the stem cells are produced from differentiated adult cells that have been induced or changed, i.e., reprogrammed, into cells capable of differentiating into tissues of all three germ layers: mesoderm, endoderm, and ectoderm. The iPSCs produced do not refer to cells as they are found in nature.II. Methods of Enriching Photoreceptors and Precursors Thereof

[0057] The disclosure features methods of enriching photoreceptor and / or photoreceptor precursor cells from a mixed population of cells. In a photoreceptor and / or photoreceptor precursor cell, the following molecules are specifically expressed: junctional adhesion molecule B (JAM-B), CD26, and / or CD133. Thus, it is possible to enrich for photoreceptor and / or photoreceptor precursor cells by sorting out of a mixed population of cells containing photoreceptor and / or photoreceptor precursor cells using the expression of at least one molecule selected from these molecular markers.

[0058] JAM-B is a protein in the family of junctional adhesion molecules (JAM), which are type I transmembrane receptors. JAMs interact with integrins, which are heterodimeric cell surface proteins that mediate cell-matrix and cell-cell interactions, and also play an important role in the assembly and maintenance of tight junctions and in the establishment of epithelial cell polarity.

[0059] CD26 is a cell surface glycoprotein with many roles including participation in T-cell activation, immunomodulation, tumorigenesis, glucose metabolism, and cell migration.

[0060] CD 133 is also a cell surface glycoprotein and is one of the most used surface markers to select and identify cancer cells and is known to be highly involved in cancer-related functions, including tumorigenesis, metastasis, chemotherapy and radiotherapy resistance, as well as apoptosis resistance.

[0061] Described herein is a method that includes contacting a mixed population of cells with a JAM-B binding agent, allowing photoreceptor cells and / or photoreceptor precursor cells to be bound by the JAM-B binding agent, and separating at least a portion of the bound photoreceptor cells and / or photoreceptor precursor cells from the mixed population of cells, thereby obtaining a population of cells enriched for photoreceptor cells and / or photoreceptor precursor cells.

[0062] The method can also include additional enrichment steps to increase the percentage of photoreceptor cells and / or photoreceptor precursor cells in the enriched population of cells. For example, the method can include contacting the enriched photoreceptor cells and / or photoreceptor precursor cells with a CD26 and / or CD133 binding agent, allowing the photoreceptor cells and / or photoreceptor precursor cells to be bound by a CD26 and / or CD133 binding agent, respectively, and separating at least a portion of the bound photoreceptor cells and / or photoreceptor precursor cells from the enriched photoreceptor cells and / or photoreceptor precursor cells to obtain a population of cells further enriched for photoreceptor cells and / or photoreceptor precursor cells.

[0063] In some embodiments, the method does not include contacting the mixed population of cells or the enriched population of cells with a CD29 binding agent or a CD15-SSEA1 binding agent. In some embodiments, the method does not include a further enrichment step using a marker that binds to a photoreceptor cell or a photoreceptor precursor cell. In some embodiments, the method does not include a further enrichment step.

[0064] Also described herein is a method of enriching photoreceptor cells and / or photoreceptor precursor cells in a mixed population of cells that includes contacting the mixed population of cells with a CD26 binding agent, allowing the photoreceptor cells and / or photoreceptor precursor cells to be bound by the CD26 binding agent, and separating at least a portion of the bound photoreceptor cells and / or photoreceptor precursor cells from the mixed population of cells, thereby obtaining a population of cells enriched for photoreceptor cells and / or photoreceptor precursor cells. In certain embodiments, the mixed population is enriched using only the CD26 binding agent and, optionally, a JAM-B binding agent. The JAM-B binding agent may be used in a subsequent or simultaneous purification step. In some embodiments, the method does not include contacting the mixed population of cells or the enriched population of cells with a CD29 binding agent or a CD15-SSEA1 binding agent. In some embodiments, the method does not include a further enrichment step using a marker that binds to a photoreceptor cell or a photoreceptor precursor cell. In some embodiments, the method does not include a further enrichment step.

[0065] Also described herein is a method of enriching photoreceptor cells and / or photoreceptor precursor cells in a mixed population of cells that includes contacting the mixed population of cells with a CD133 binding agent, allowing the photoreceptor cells and / or photoreceptor precursor cells to be bound by the CD133 and separating at least a portion of the bound photoreceptor cells and / or photoreceptor precursor cells from the mixed population of cells, thereby obtaining a population of cells enriched for photoreceptor cells and / orphotoreceptor precursor cells. In certain embodiments, the mixed population is enriched using only the CD 133 binding agent and a JAM-B binding agent. The JAM-B binding agent may be used in a subsequent or simultaneous purification step. In some embodiments, the method does not include contacting the mixed population of cells or the enriched population of cells with a CD29 binding agent or a CD15-SSEA1 binding agent. The JAM-B binding agent may be used in a subsequent or simultaneous purification step.

[0066] In some embodiments, the method enriches for photoreceptor cells. In some embodiments, the photoreceptor cells are cone photoreceptor cells.

[0067] In some embodiments, the method enriches for photoreceptor precursor cells. In some embodiments, the photoreceptor precursor cells are cone photoreceptor precursor cells.

[0068] The molecular marker used as an indication for photoreceptor and / or photoreceptor precursor cells may, in some embodiments, be a single member selected from the group consisting of JAM-B, CD26, and CD133, or the marker may, in some embodiments, be any combination of two or three of these molecular markers. In some embodiments, the molecular marker is JAM-B. In some embodiments, the molecular markers are JAM-B and CD26. In some embodiments, the molecular markers are JAM-B and CD 133. In some embodiments, the molecular markers are JAM-B, CD26, and CD133.

[0069] Sorting of one or more photoreceptor (e.g., cones or rods) and / or photoreceptor precursor cells from a mixed population of cells by the expression of at least one molecular marker selected from the group consisting of JAM-B, CD26, and CD133 can be performed, for example, by sorting cells in which at least one of the molecular markers is expressed.

[0070] The expression of the molecular marker can be detected by any available method. Examples of typical detection methods include a method comprising measuring the expression of the gene(s) that encode the at least one molecular marker by RT-PCR, and a method comprising detecting the presence of the at least one molecular marker using a substance that specifically binds to the at least one molecular marker. All of the molecular markers listed above are a surface protein of the photoreceptor and / or photoreceptor precursor cell. Because the molecular marker can be detected with cells remaining viable, the method preferably comprises detecting a molecular marker using an agent that specifically binds to the marker.

[0071] The type of substance that specifically binds to the molecular marker is not particularly limited. Examples of JAM-B, CD26, and / or CD133 binding agents include antibodies, nanobodies, and aptamers. In some embodiments, the agent is an antibody or a fragment thereof. The antibody may be either a polyclonal antibody or a monoclonal antibody. Examples of antibody fragment include Fab fragments, F(ab)2 fragments, and ScFv fragments.When expression of two or more types of molecular markers are used to detect photoreceptor and / or photoreceptor precursor cells, agents that specifically bind to respective markers may be used in combination.

[0072] The antibody for specifically recognizing a molecular marker may be a commercially available antibody or may be prepared by a well-known method. There are well-known methods for preparing antibodies. For example, a polyclonal antibody can be prepared by immunizing a non-human animal with purified molecular markers or their partial peptides and obtaining serum of the animal in accordance with an ordinary method. A monoclonal antibody can be obtained from a hybridoma prepared by fusing spleen cells from an immunized animal with myeloma cells.

[0073] In some embodiments, the JAM-B binding agent is an antibody or fragment thereof. In some embodiments, the JAM-B binding agent is a nanobody. In some embodiments, the JAM- B binding agent is an aptamer. In some embodiments, the CD26 binding agent is an antibody or fragment thereof. In some embodiments, the CD26 binding agent is a nanobody. In some embodiments, the CD26 binding agent is an aptamer. In some embodiments, the CD133 binding agent is an antibody or fragment thereof. In some embodiments, the CD133 binding agent is a nanobody. In some embodiments, the CD133 binding agent is an aptamer.

[0074] To facilitate the detection of cells bound to an agent that specifically binds to a molecular marker, the agent is preferably labeled with a labeling substance. The labeling substance is not particularly limited. Examples of labeling substances include fluorescent materials, magnetic materials, radioactive materials, chemiluminescent materials, enzymes, biotin, and streptavidin. The agent that specifically binds to a molecular marker may be indirectly labeled. For example, a pre-labeled antibody (e.g., a secondary antibody) that specifically binds to the agent (e.g., an antibody) may be used.

[0075] The method for recognizing cells bound to an agent that specifically binds to a molecular marker to sort the cells is not particularly limited, and any method known and hereafter developed may suitably be selected. For example, the method for recognizing / sorting cells can be selected according to the type of labeling substance for use. Typical methods for recognizing / sorting cells include fluorescence-activated cell sorting (FACS), magnetic-activated cell sorting (MACS), cell panning, and affinity chromatography. The mode of FACS is not particularly limited. For example, FACS may be either a droplet-charge mode or a cell-capturing mode.

[0076] As described above, photoreceptor and / or photoreceptor precursor cells can be enriched by sorting photoreceptor and / or photoreceptor precursor cells from a mixed populationof cells containing one or more photoreceptor and / or photoreceptor precursor cells using a molecular marker. Following any of the methods described herein, it is possible to obtain a cell population enriched for photoreceptor and / or photoreceptor precursor cells at a significantly high concentration. Thus, the methods for enriching photoreceptor and / or photoreceptor precursor cells described herein can also be used to enrich for photoreceptor and / or photoreceptor precursor cells, wherein the enriched population of cells comprises at least about 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, and / or 90% photoreceptor and / or photoreceptor precursor cells. In some embodiments, the enriched population of cells comprises at least about 60-90%, 65-85%, and / or 70-80% photoreceptor and / or photoreceptor precursor cells.

[0077] The photoreceptor and / or photoreceptor precursor cells obtained by the methods described herein can be further cultured. The further culture can be performed, for example, in a differentiation-induction medium so that the photoreceptor and / or photoreceptor precursor cells become more mature. The further culture can be performed under conditions suitable for proliferation of photoreceptor and / or photoreceptor precursor cells, or for maintenance of the viability of the cells.Mixed Population of Cells

[0078] The disclosure features methods of enriching photoreceptor and / or photoreceptor precursor cells from a mixed population of cells. In some embodiments, the mixed population of cells comprises differentiated human induced pluripotent stem (iPS) or embryonic stem (ES) cells.

[0079] The ES cells of some embodiments of the disclosure can be obtained using well- known cell culture methods. For example, human ES cells can be isolated from human blastocysts. Human blastocysts are typically obtained from human in vivo preimplantation embryos or from in vitro fertilized (IVF) embryos. Alternatively, a single cell human embryo can be expanded to the blastocyst stage. For the isolation of human ES cells the zona pellucida is removed from the blastocyst and the inner cell mass (ICM) is isolated by a procedure in which the trophectoderm cells are lysed and removed from the intact ICM by pipetting. The ICM is then plated in a tissue culture flask containing the appropriate medium which enables its outgrowth. Following 9 to 15 days, the ICM derived outgrowth is dissociated into clumps either by mechanical dissociation or by enzymatic digestion. The cells are then re-plated on a fresh dish. Colonies demonstrating undifferentiated morphology are individually selected by micropipette,mechanically dissociated into clumps, and re-plated. Resulting ES cells are then routinely split every 4-7 days. For further details on methods for preparation of human ES cells see Reubinoff et al. (2000) Nat Biotechnol 18(5): 559; Thomson et al., (U.S. Pat. No. 5,843,780; (1998) Science 282: 1145; (1998) Curr. Top. Dev. Biol. 38: 133; (1995) Proc. Natl Acad. Sci. USA 92: 7844); Bongso c / o / . (1989) Hum Reprod '. 706, and Gardner et al. (1998) Fert / 7. Steril. 69: 84.

[0080] It will be appreciated that commercially-available ES cells can also be used according to some embodiments of the disclosure. Human ES cells can be purchased from the NIH human ES cells registry (www.grants(dot)nih (dot)gov / stem cells / registry / current(dot)htm) or from other hESC registries. Non-limiting examples of commercially available ES cell lines are HAD-C102, ESI, BG01, BG02, BG03, BG04, CY12, CY30, CY92, CY10, TE03, TE32, CHB-4, CHB-5, CHB-6, CHB-8, CHB-9, CHB-10, CHB-11, CHB-12, HUES 1, HUES 2, HUES 3, HUES 4, HUES 5, HUES 6, HUES 7, HUES 8, HUES 9, HUES 10, HUES 11, HUES 12, HUES 13, HUES 14, HUES 15, HUES 16, HUES 17, HUES 18, HUES 19, HUES 20, HUES 21, HUES 22, HUES 23, HUES 24, HUES 25, HUES 26, HUES 27, HUES 28, CyT49, RUES3, WA01, UCSF4, NYUES1, NYUES2, NYUES3, NYUES4, NYUES5, NYUES6, NYUES7, UCLA 1, UCLA 2, UCLA 3, WA077 (H7), WA09 (H9), WA13 (H13), WA14 (H14), HUES 62, HUES 63, HUES 64, CT1, CT2, CT3, CT4, MA135, Eneavour-2, WIBR1, WIBR3, WIBR4, WIBRS, WIBR6, HUES 45, Hl, H9, Shef 1.3, Shef 3, Shef 6, BJNheml9, BJNhem20, and SA001. ES cells may be a cellular source for photoreceptors and photoreceptor precursor cells.

[0081] Induced pluripotent stem cells (iPSCs) can be generated by genetic manipulation of somatic cells, e.g., by retroviral transduction of somatic cells such as fibroblasts, hepatocytes, or gastric epithelial cells with transcription factors such as Oct-3 / 4, Sox2, c-Myc, and KLF4 (Yamanaka (2007) Cell Stem Cell. l(l):39-49; Aoi T, et al. (2008) Science 321(5889):699-702; Park et al. (200 ) Nature 451 : 141-146; Takahashi et al. (2007) Cell 131 :861-872). Other embryonic-like stem cells can be generated by nuclear transfer to oocytes, fusion with ES cells or nuclear transfer into zygotes if the recipient cells are arrested in mitosis. In addition, iPSCs may be generated using non-integrating methods e.g., using small molecules or RNA. iPSCs may be a cellular source of photoreceptors and photoreceptor precursor cells.

[0082] In some embodiments, the mixed population of cells comprises human retinal organoid (RO) cells. An organoid is a self-organized tissue induced from stem cells that is able to form three-dimensional structures with a variety of cell types. Mature ROs have all of the major cell types and structures forming the retina. Currently, human ES cells, iPSCs, retinal progenitor cells (RPCs), mesenchymal stem cells, and other types of stem cells have been used to cultivate ROs with multi-cell retinal laminar structure and ultrastructure. Using methods known in the art,a human RO can be generated, such as those described in e.g., Nakano et al. Cell stem cell 10.6 (2012): 771-785; Yu, et al. Advanced Drug Delivery Reviews 197 (2023): 114842; Sun and Liang. Biofabrication, 15 (3).” (2023); Ma et al. Experimental Eye Research (2024): 110068; and Li et al. JoVE (Journal of Visualized Experiments) 202 (2023): e66246, each of which is incorporated herein in its entirety as it pertains to methods of making ROs, and the methods described herein can be used on human RO cells.Ill Methods of Treating Retinal Disorders

[0083] Using the above described methods, a population of photoreceptor and / or photoreceptor precursor cells can be obtained. Furthermore, the photoreceptor and / or photoreceptor precursor cells enriched by using the molecular markers described above can be used in the treatment of retinal disorders. Thus, there can be provided a pharmaceutical composition containing a population of photoreceptor and / or photoreceptor precursor cells for treatment of retinal disorders. This pharmaceutical composition may further contain various components, scaffold materials, carriers, etc., to aid the maintenance and proliferation of the photoreceptor and / or photoreceptor precursor cells, or to aid application of the composition to the affected area.

[0084] Examples of the components to aid the maintenance and / or proliferation of the cells include components used for media, such as carbon sources, nitrogen sources, vitamins, minerals, salts, and various cytokines. Examples of the scaffold materials to aid the application of the composition to the affected area include collagen, polylactic acid, hyaluronic acid, cellulose, and derivatives thereof. These components and scaffold materials may be used in a combination of two or more. The pharmaceutical composition may be in the form of an injectable aqueous solution (e.g., physiological saline; physiological buffers, such as PBS; and isotonic solutions containing glucose and / or other adjuvants) to which photoreceptor and / or photoreceptor precursor cells have been added.

[0085] A cellular population of photoreceptor and / or photoreceptor precursor cells can be obtained by culturing photoreceptor and / or photoreceptor precursor cells obtained using the enriched cells obtained using the above described molecular markers on a suitable carrier (e.g., a polymer membrane). The carrier is not particularly limited, and examples of the carrier include biopolymers, such as collagen, atelocollagen, alkali-treated collagen, gelatin, keratin, hyaluronic acid, glycosaminoglycan (e.g., chondroitin sulfate, dermatan sulfate, hyaluronic acid, heparan sulfate, heparin, and keratan sulfate), proteoglycan, alginic acid, chitosan, polyamino acid(polylactic acid), and cellulose; and temperature-responsive polymers, such as (meth)acrylamide compounds, N-(or N,N-di)alkyl substituted (meth)acrylamide derivatives, vinyl ether derivatives, and copolymers thereof.

[0086] A protectant or similar component may optionally be added to the photoreceptor and / or photoreceptor precursor cells to cry opreserve the cells. Examples of the protectant include glycerol, dimethyl sulfoxide (DMSO), propylene glycol, and acetamide. To maintain the safety of the cells as a graft, the cells can be subjected to heat treatment and / or radiation treatment, or the like.

[0087] Retinal disorders that can be treated in accordance with the populations of cells obtained from the enrichment methods described herein include, but are not limited to, inherited retinal degenerations (IRDs), age-related macular degeneration (AMD), Stargardt disease, myopic macular degeneration (MMD), sub-macula hemorrhage, diabetic retinopathy (DR), diabetic macular edema (DME), and uveitis. AMD may be classified as wet or neovascular AMD or dry AMD (also called geographic atrophy (GA)). For example, in some embodiments, a population of cells is obtained from differentiated iPS or ES cells or RO cells wherein at least 80% of the population of cells are photoreceptor and / or photoreceptor precursor cells. Such a population of cells can be obtained using any of the methods described herein and the population of cells are administered with a method for treating a retinal disorder.

[0088] In some embodiments, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of the population of cells are photoreceptor and / or photoreceptor precursor cells.

[0089] In some embodiments, the photoreceptor and / or photoreceptor precursor cells express JAM-B on their surface. In some embodiments, the photoreceptor and / or photoreceptor precursor cells express CD26 on their surface. In some embodiments, the photoreceptor and / or photoreceptor precursor cells express CD133 on their surface.Pharmaceutical Compositions and Routes of Administration

[0001] Preferably the cells are mixed with or seeded onto a pharmaceutically acceptable carrier prior to administration. As used herein, the term “pharmaceutically acceptable” refers to those compounds, materials, compositions, and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio. Such formulations can be prepared using techniques well known in the art. See, e.g., U.S. Patent Application 2003 / 0180289; and Remington: The Science and Practice of Pharmacy, Alfonso R. Gennaro,editor, 20th ed. Lippincott Williams & Wilkins: Philadelphia, PA, 2000.

[0002] The carrier may be a solid or a liquid, or both (e.g., hydrogels), and can be formulated with the cells as a unit-dose formulation. In some embodiments the cells are provided as a suspension in the carrier to reduce clumping of the cells. In other embodiments cells are seeded onto a biodegradable scaffold or matrix.

[0090] The cell therapy compositions described herein can comprise one or more carriers and / or excipients. Pharmaceutically acceptable carriers include any of the standard pharmaceutical carriers, such as a phosphate buffered saline (PBS) solution, water, emulsions (e.g, such as an oil / water or water / oil emulsions), and various types of wetting agents. The compositions also can include stabilizers and preservatives. For examples of carriers, stabilizers and adjuvants, see, e.g., Adeboye Adejare, Remington: The Science and Practice of Pharmacy (23d ed. 2020). Pharmaceutically acceptable carriers include buffers, solvents, dispersion media, coatings, isotonic and absorption delaying agents, and the like, that are compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is known in the art.

[0091] In some embodiments, cells are mixed with a suitable gel for administration. Suitable gels that may be used in the present disclosure include, but are not limited to, agars, collagen, fibrin, hydrogels, etc. Besides gels, other support compounds may also be utilized in the present disclosure. Extracellular matrix analogs, for example, may be combined with support gels to optimize or functionalize the gel. One or more growth factors may also be introduced into the cell suspensions.

[0092] Formulations of the disclosure include those for parenteral administration (e.g, subcutaneous, intramuscular, intradermal, intravenous, intraarterial, and intraperitoneal injection) by injection or implantation. In some embodiments, administration is carried out intravascularly, either by simple injection, or by injection through a catheter positioned in a suitable blood vessel. In some embodiments, administration is carried out by ‘infusion,’ whereby compositions are introduced into the body through a vein (e.g, the portal vein). In another embodiment, administration is carried out as a graft to an organ or tissue to be augmented as discussed above, e.g., the retina of the eye.

[0093] A “biodegradable scaffold or matrix” is any substance not having toxic or injurious effects on biological function and is capable of being broken down into is elemental components by a host. Preferably, the scaffold or matrix is porous to allow for cell deposition both on and in the pores of the matrix. Such formulations described herein can be prepared by supplying at leastone cell population to a biodegradable scaffold to seed the cell population on and / or into the scaffold. The seeded scaffold may then be implanted in the body of a recipient subject.

[0094] In some embodiments, cells are administered by injection of the cells (e.g., in a suitable carrier) directly into the tissue of a subject. For example, cells may be injected into the eye, retina, or intravitreal space. In some embodiments, the cells may be injected transvitreally or via a suprachoroidal route.

[0095] Cells may also be delivered systemically. See, e.g., the “Edmonton protocol,” an established delivery method, where cells are infused into a patient's portal vein (Shapiro et al. (2000) TV Engl J Med 343:230-238).

[0096] According to some embodiments, the cells administered to the subject may be syngeneic (i.e., genetically identical or closely related, so as to minimize tissue transplant rejection), allogeneic (i.e., from a non-genetically identical member of the same species) or xenogeneic (i.e., from a member of a different species), as above, with respect to the subject being treated, depending upon other steps such as the presence or absence of encapsulation or the administration of immune suppression therapy of the cells. Syngeneic cells include those that are autogeneic (i.e., from the subject to be treated) and isogeneic (i.e., a genetically identical but different subject, e.g., from an identical twin). Cells may be obtained from, e.g., a donor (either living or cadaveric) or derived from an established cell strain or cell line. As an example of a method that can be used to obtain cells from a donor (e.g., a potential recipient of a bioscaffold graft), standard biopsy techniques known in the art may be employed. Alternatively, cells may be harvested from the subject, expanded / selected in vitro, and reintroduced into the same subject (i.e., autogeneic).

[0097] In some embodiments, cells are administered in a therapeutically effective amount. The therapeutically effective dosage of cells will vary somewhat from subject-to- subject, and will depend upon factors such as the age, weight, and condition of the subject and the route of delivery. Such dosages can be determined in accordance with procedures known to those skilled in the art.

[0098] In further embodiments, if desired or necessary, the subject may be administered an agent for inhibiting transplant rejection of the administered cells, such as rapamycin, azathioprine, corticosteroids, cyclosporin, and / or FK506, in accordance with known techniques. See, e.g., R. Caine, U.S. Patent Nos. 5,461,058; 5,403,833; and 5,100,899; see also U.S. Patent Nos. 6,455,518; 6,346,243; and 5,321,043. Some embodiments use a combination of implantation and immunosuppression, which minimizes graft rejection. The implantation may be repeated as needed to create an adequate mass of transplanted tissue.EXAMPLES

[0099] Below are examples of specific embodiments for carrying out the present invention. The examples are offered for illustrative purposes only and are not intended to limit the scope of the present invention in any way. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperatures, etc.), but some experimental error and deviation should, of course, be allowed for.

[0100] The practice of the present invention will employ, unless otherwise indicated, conventional methods of protein chemistry, biochemistry, recombinant DNA techniques and pharmacology, within the skill of the art. Such techniques are explained fully in the literature. See, e.g., T.E. Creighton, Proteins: Structures and Molecular Properties (W.H. Freeman and Company, 1993); A.L. Lehninger, Biochemistry (Worth Publishers, Inc., current addition); Sambrook, et al., Molecular Cloning: A Laboratory Manual (2nd Edition, 1989); Methods In Enzymology (S. Colowick and N. Kaplan eds., Academic Press, Inc.); Remington’s Pharmaceutical Sciences, 18th Edition (Easton, Pennsylvania: Mack Publishing Company, 1990); Carey and Sundberg Advanced Organic Chemistry 3rd Ed. (Plenum Press) Vols A and B(1992).Example 1: Methods for Enriching Photoreceptors and Photoreceptor Precursor Cells

[0001] This example describes methods of enriching photoreceptor cells and photoreceptor precursor cells from a mixed population of cells.Materials and Methods iPSC cryo-preservation

[0101] Human iPSC lines were stored frozen in 1 mL cryoprotectant (CryoStor® CS10, STEMCELL Technologies, Cat. Nr. 07959) in cryogenic storage vials (cryovials, Fisher Scientific, Cat. Nr. 11787939) inside a nitrogen tank. For freezing cells in culture in a well of a 6- well plate, the expended medium was first removed from a well at 70-90% confluency. Then, cells were washed with 1 mL room temperature (RT) sterile Dulbecco’s PBS (DPBS; CTS™, without calcium chloride, without magnesium chloride, ThermoFisher Scientific, Cat. Nr. A1285601) and incubated for 6 minutes at RT with 1 mL Gentle Cell Dissociation Reagent (GCDR, STEMCELL Technologies, Cat. Nr. 100-0485). Afterwards, Gentle Cell Dissociation Reagent (GCDR) was removed, cells were resuspended in 3 mL CS10, and the volume distributed equally to 3 cryovials. Cryovials were immediately deposited inside a CoolCell® container (Coming, Cat. Nr. 432000) and transferred to a -80 °C freezer. Containers were kept there for a minimum of 12 hours and a maximum of 72 hours period during which vials weretransferred to a nitrogen tank for long-term storage. All steps involving manipulation of the cells were performed under sterile conditions inside microbiological safety cabinets (MB SC) with laminar flow. iPSC thawing

[0102] For thawing, cryovials were collected from the nitrogen tank and transferred to the tissue culture room on dry ice. Once all reagents were ready, cryovials were held inside the fist for as long as required until only a small ball of ice was visible inside them. At this point, cryovials were sprayed with 70% ethanol and transferred to the MBSC. There, vials were opened and their contents were transferred to 15 mL conical tubes. Immediately after, 6 mL RT mTeSR medium (refers to the mix of the two components in the mTeSR™ Plus cGMP Kit (STEMCELL Technologies, Cat. Nr. 100-0276): mTeSR™ Plus Basal Medium (400 mL) and mTeSR™ Plus 5X Supplement (100 mL)) was added dropwise to the cells. Next, cells were centrifuged at 300 g for 4 minutes at RT. Then, the supernatant was removed, the cell pellet was resuspended in 1 mL mTeSR supplemented with 10 pM ROCK inhibitor (mTeSR+ROCKi; Y-27632, STEMCELL Technologies, Cat. Nr. 72307), and cells were transferred to 6-well tissue culture plates precoated with CTS™ Vitronectin (CTS-VTN, ThermoFisher Scientific, Cat. Nr. CTS279S3) prefilled with 2 mL mTeSR+ROCKi. Plate coating was performed by mixing CTS-VTN with DPBS at a 1 :90 ratio, adding 1 mL of this mix per well of a 6-well plate, and incubating the plates with filled wells for 1 hour at RT. After cells were seeded, plates were transferred to and kept in 5% CO2 incubators at 37 °C. iPSC maintenance and passaging

[0103] The morning after iPSC thawing, expended medium was removed and replaced with 2 mL fresh mTeSR at RT. Media changes took place daily from Monday to Thursday. On Fridays, cells were either passaged or fed with 4 mL fresh mTeSR. No media changes took place on Saturdays and Sundays. For passaging, the expended medium was removed, and cells were washed with 1 mL DPBS, incubated with 1 mL GCDR for 6 min, and re-suspended in 1 mL mTeSR+ROCKi. Immediately after, cells were transferred at the desired concentration to wells of 6-well plates pre-coated with 1 mL VTN-CTS and pre-filled with 2 mL mTeSR+ROCKi. Expended medium was removed and fresh mTeSR at RT was added the following morning. iPSC differentiation into human retinal organoids (HROs)

[0104] iPSCs were differentiated into human retinal organoids (HROs) following a previously published protocol (Reichman et al., Proceedings of the National Academy of Sciences 111.23 (2014): 8518-8523) with minor modifications. In brief, after dissociation with GCDR, iPSCs were seeded at a 1 :30 dilution in 60-mm tissue culture-treated cell culture dishes (Coming, Cat. Nr. 430166) pre-coated with VTN-CTS (4 mL per dish) and pre-filled with 4 mL mTeSR + ROCKi. After 72 hours, expanded medium was removed and 4 mL fresh mTeSR at RT was added. Once the cells reached a confluency of 70-80% (usually 5 days after seeding), expanded medium was removed and replaced with 4 mL Essential 6 medium (E6, ThermoFisher Scientific, Cat. Nr. A4238501). One day after, expended E6 was removed and replaced with fresh RT E6. The following day (referred to as Day 2 (D2)), expended E6 was removed and replaced with 4 mL neuroectoderm induction medium (NIM, made by supplementing E6 with CTS™ N-2 Supplement, ThermoFisher Scientific, Cat. Nr. A1370701, at a 1 : 100 dilution). From this timepoint onwards, expended NIM was removed and 4 mL fresh NIM were added to the culture every Monday, Wednesday, and Friday until D28.

[0105] On D28, iPSC-derived optic vesicles (OVs) were identified under a microscope by their characteristic round shape with dark edges and slightly opaque core. OVs were dissected by hand under sterile conditions inside a MB SC with laminar flow using a 23 -gauge syringe needle onto a 1 mL syringe, as previously described. After dissection, floating OVs were transferred with a 200 pL pipette to a well of a Costar® ultra-low attachment 6-well plate (Coming, Cat. Nr. 3471) pre-filled with 4 mL retinal maturation medium (RMM; components: Gibco™ CTS™ KnockOut™ DMEM / F- 12 (ThermoFisher Scientific, Cat. Nr. A1370801), CTS™ GlutaMAX™ I Supplement (ThermoFisher Scientific, Cat. Nr. A1286001) at a 1 : 100 dilution, MEM non- essential amino acids solution (100X) (ThermoFisher Scientific, Cat. Nr. 11140050) at a 1 : 100 dilution, CTS™ B-27™ Supplement XenoFree (50X) (ThermoFisher Scientific, Cat. Nr. A5047501) at a 1 :50 dilution, and penicillin-streptomycin (10,000 U / mL) (ThermoFisher Scientific, Cat. Nr. 15140122) at a 1 : 1000 dilution) supplemented with recombinant fibroblast growth factor-2 (FGF-2, R&D Systems, Cat. Nr. 233-GMP-025) at a concentration of 10 ng / mL.

[0106] Half of the FGF-2-supplemented RMM (2 mL) was removed and replaced with 2 mL of fresh FGF-2-supplemented RMM every Monday, Wednesday, and Friday until D34. From D35 onwards, half of the medium (2 mL) was removed and replaced with 2 mL fresh RMM on Mondays, Wednesdays, and Fridays until the timepoint of analysis. All steps were performed allowing the temperature of the fresh medium (stored at 4 °C) to first equilibrate to RT and paying special attention not to aspirate and discard the OVs / HROs.HRO dissociation into single cell suspension

[0107] HROs were dissociated into single cell suspensions using papain. In brief, 89 units papain per mL papain-activating solution (PAS, components: 1.1 mM ethylenediaminetetraacetic Acid (EDTA, Sigma-Aldrich, Cat. Nr. E6758) and 5.5 mM L-Cysteine (Sigma-Aldrich, Cat. Nr. C7477) in sterile distilled water) supplemented with P-mercaptoethanol (Sigma-Aldrich, Cat. Nr. M3 148) at a final concentration of 0.066 mM were incubated for 20 min at 37 °C in a heating block. In the meantime, HROs were collected from the culture plates, transferred to 1.5 mL microcentrifuge tubes (10-15 per tube), and washed twice with 1 mL Ringer’s buffer (components: 125 mM NaCl (Sigma-Aldrich, Cat. Nr. S7653), 3.6 mM KCl (Sigma-Aldrich, Cat. Nr. P5405), 1.18 mM MgCh (hexahydrate) (Sigma-Aldrich, Cat. Nr. M2393), 22.6 mM NaHCCh (Sigma-Aldrich, Cat. Nr. S6297), 0.02 mM NaH2PO4(Sigma-Aldrich, Cat. Nr. S9638), 0.028 mM Na2HPO4 (Sigma-Aldrich, Cat. Nr. S7907), 1.2 mM Na2SO4 (Sigma-Aldrich, Cat. Nr.238597), 10 mM glucose (Sigma-Aldrich, Cat. Nr. G7021), and 0.54 M EDTA Na2(Sigma- Aldrich, Cat. Nr. E6635) in sterile distilled water). After the Papain and PAS incubation was completed, the mix was diluted 1 : 10.4 in Ringer’s buffer and HROs transferred to it (50 pL Papain, PAS, and Ringer’s per HRO) for incubation at 37 °C on a shaker at 300 RPM. For the following hour, HROs were pipetted up and down 15-20 times with a Pl 000 pipette every 20 minutes. Afterwards, the enzymatic activity of papain was inhibited by diluting 1 :5 the totality of the cell suspension in RMM. Additionally, to minimize cell clumping as a consequence of free- floating genomic DNA from dead cells, the diluted cell suspension was incubated for 5 min at RT with DNAse I (Merck, Cat. Nr. #D4263-1VL) at a concentration of 20 pL / mL.Immunolabeling of HRO-derived single cell suspensions

[0108] After the incubation with DNAse I was completed, cell suspensions were filtered through the cell strainer cap of round-bottom 5 mL polystyrene test tubes (Fisher Scientific, Cat. Nr. 0877123) and the flow-through was centrifuged at 240 G for 4 min. Afterwards, cells were resuspended in MACSQuant Tyto Running Buffer (MQT Buffer, Miltenyi Biotec, Cat. Nr. 130- 107-207) and dispensed in wells of 96-well conical (V) bottom plates at 100 pL per well. Then, one of the primary antibodies described in TABLE 1 was added per condition (dilutions: CD 133, 1 :50; CD26, 1 :5; and JAM-B, 1 :50) and cells were incubated on ice for 30 min. After the incubation period, wells were topped up with 150 pL MQT buffer each, plates were centrifuged at 240 G for 4 min and supernatants were removed. An additional washing step was then performed using 250 pL MQT buffer, followed by centrifugation of the plates at 240 G for 4 min. At this stage, CD133-stained and CD26-stained cells were resuspended in 1 mL MQTbuffer and sorted in a MACSQuant Tyto. Meanwhile, before sorting this way, JAM-B-labelled cells were incubated with an APC-conjugated goat anti-mouse antibody (TABLE 2) at a 1 :2000 dilution in MQT buffer on ice for 30 min, and thereafter washed twice with MQT buffer prior to resuspension in 1 mL MQT buffer for sorting (as described above). Non-stained cells were kept as controls (unstained). Experiments were performed using biological triplicates.Cell sorting and flow cytometry analysis

[0109] Prior to sorting, at least 10,000 “unstained” control and 10,000 stained (“pre-sort”) cells were run through a flow cytometer (Aurora Northern Lights, Cytek) to confirm absence of positive cells in the absence of labelling and to assess pre-sort fractions of mCherry -positive and marker-positive (CD133, CD26, or JAM-B) cells. The flow cytometer was operated using standard settings (Forward Scattering (FSC): 25, Side Scattering (SSC): 131, Side Scattering- Blue (SSC-B): 145). In preparation for sorting, MACSQuant Tyto cartridges were primed with 500 pL MQT buffer following the manufacturer’s instructions and the remaining volume of cell suspension (after analysis of the “pre-sort” fraction) was loaded into the primed MACSQuant Tyto cartridge for sorting in the MACSQuant Tyto using standard settings (SSC: 500 V; BSB: 580 V; Noise threshold: 21,20). Channel voltages were adjusted to maximize resolution between the positive and negative population. Sorted cells were resuspended in 200 pL MQT buffer from their corresponding chamber in the cartridge using a 200 pL pipette and transferred to a 5 mL polystyrene tube for measurement in the same flow cytometer with the same settings as used for the “unstained” and “pre-sort” samples. Flow cytometry standard (FCS) output files were exported and analyzed offline using FlowJo (vl0.9.0, FlowJo LLC).TABLE 1. List of primary antibodiesTABLE 2. List of secondary antibodiesResults

[0110] The reporter human iPS cell lines herein referred to as Crx-mCherry and Car-mCherry express the fluorescent reporter protein mCherry under the control of minimal promoters for the murine genes Crx (Cone-rod homeobox) and Arr 3 (Arrestin-3; also known as retinal cone arrestin-3). In HROs, mCherry-positive cells derived from Crx-mCherry iPSCs were considered to have committed to the photoreceptor fate and to be either photoreceptor precursors or mature photoreceptors - including both rods and cones. Meanwhile, the mCherry-positive cells that emerge when differentiating the Car-mCherry cell line into HROs were considered to be either cone photoreceptor precursors or mature cone photoreceptors.

[0111] Using these cell lines, antibodies that can be used to immunolabel photoreceptorspecific and / or photoreceptor precursor-specific antigens were identified and used to subsequently enrich such populations. Specifically, antibodies recognizing epitopes of CD26 (dipeptidyl-peptidase IV; FIGs. 1A-1F), CD133 (prominin-1; FIGs. 2A-2F), and JAM-B (junctional adhesion molecule B; FIGs. 3A-3F), allowed for a significant enrichment of mCherry-positive cells using the MACSQuant Tyto device (Miltenyi Biotec) for enrichment and fluorescence-activated cell sorting (FACS) for quantification. In particular, the fraction of immunolabelled cells before and after enrichment was determined using a Cytek Aurora spectral flow cytometer with standard settings. Statistical significance was calculated in GraphPad Prism (version 10.2.3) using a Student’s t-test with Welch’s correction comparing the ‘Pre-sort’ and ‘Enriched’ conditions. All experiments were conducted in three independent differentiation experiments when HROs were an age of 120 to 150 days.

[0112] In the Crx-mCherry line, usage of the CD26 marker allowed enrichment of the fraction of CD26 / mCherry double-positive cells from 33.5 ± 2.2% (pre-sort) to 89.0 ± 1.8 (enriched; -value < 0.0001) (values expressed as average ± S.E.M.). Meanwhile, in the Car- mCherry line, the respective values were 28.8 ± 3.9% (pre-sort) and 81.3 ± 3.8% (enriched; P- value = 0.007). In comparison, the CD133 marker enabled the enrichment of CD133 / mCherry double-positive cells from 49.0 ± 8.2% to 96.8 ± 0.6% and from 36.0 ± 4.5% to 87.4 ± 1.4% in the Crx-mCherry and Car-mCherry cell lines, respectively (P = 0.0276, Crx-mCherry; P = 0.0043, Car-mCherry). Finally, using JAM-B, the fraction of JAM-B / mCherry double-positive cells was enriched from 62.4 ± 1.9% to 92.2 ± 1.3% in the Crx-mCherry line (P-value = 0.0004), and from 41.6 ± 6.3% to 94.8 ± 0.9% in the Car-mCherry line (P-value = 0.0124). Hence, these three markers (CD26, CD 133, and JAM-B) were demonstrated to be suitable for the enrichment of photoreceptor cells from human retinal organoids.Equivalents and Scope

[0113] Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments in accordance with the invention described herein. The scope of the present invention is not intended to be limited to the above Description, but rather is as set forth in the appended claims.

[0114] Claims or descriptions that include “or” between one or more members of a group are considered satisfied if one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process unless indicated to the contrary or otherwise evident from the context. The invention includes embodiments in which exactly one member of the group is present in, employed in, or otherwise relevant to a given product or process. The invention includes embodiments in which more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process.

[0115] It is also noted that the term “comprising” is intended to be open and permits but does not require the inclusion of additional elements or steps. When the term “comprising” is used herein, the term “consisting of’ is thus also encompassed and disclosed.

[0116] Where ranges are given, endpoints are included. Furthermore, it is to be understood that unless otherwise indicated or otherwise evident from the context and understanding of one of ordinary skill in the art, values that are expressed as ranges can assume any specific value or subrange within the stated ranges in different embodiments of the invention, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise.

[0117] Throughout the description, where compositions are described as having, including, or comprising specific components, or where processes are described as having, including, or comprising specific process steps, it is contemplated that compositions of the present invention also consist essentially of, or consist of, the recited components, and that the processes of the present invention also consist essentially of, or consist of, the recited processing steps. Further, it should be understood that the order of steps or order for performing certain actions are immaterial so long as the invention remains operable. Moreover, two or more steps or actions may be conducted simultaneously.

[0118] All cited sources, for example, references, publications, databases, database entries, and art cited herein, are incorporated into this application by reference, even if not expressly stated in the citation. In case of conflicting statements of a cited source and the instant application, the statement in the instant application shall control.

[0119] Section and table headings are not intended to be limiting.OTHER EMBODIMENTS

[0120] It is to be understood that the words which have been used are words of description rather than limitation, and that changes may be made within the purview of the appended claims without departing from the true scope and spirit of the invention in its broader aspects.

[0121] While the present invention has been described at some length and with some particularity with respect to the several described embodiments, it is not intended that it should be limited to any such particulars or embodiments or any particular embodiment, but it is to be construed with references to the appended claims so as to provide the broadest possible interpretation of such claims in view of the prior art and, therefore, to effectively encompass the intended scope of the invention.

Claims

CLAIMS1. A method of enriching photoreceptor cells and / or photoreceptor precursor cells from a mixed population of cells, the method comprising: contacting the mixed population of cells with a JAM-B binding agent, allowing the photoreceptor cells and / or photoreceptor precursor cells to be bound by the JAM-B binding agent, and separating at least a portion of the bound photoreceptor cells and / or photoreceptor precursor cells from the mixed population of cells, thereby obtaining a population of cells enriched for photoreceptor cells and / or photoreceptor precursor cells.

2. The method of claim 1, the method further comprising performing a second enrichment by: contacting the enriched photoreceptor cells and / or photoreceptor precursor cells with a CD26 and / or CD133 binding agent, allowing the photoreceptor cells and / or photoreceptor precursor cells to be bound by the CD26 and / or CD133 binding agent, respectively, and separating at least a portion of the bound photoreceptor cells and / or photoreceptor precursor cells from the enriched photoreceptor cells and / or photoreceptor precursor cells to obtain a population of cells further enriched for photoreceptor cells and / or photoreceptor precursor cells.

3. The method of claim 1 or 2, wherein the JAM-B binding agent is an antibody or a nanobody.

4. The method of claim 2 or 3, wherein the CD26 and / or CD133 binding agent is an antibody or a nanobody.

5. The method of any one of claims 1-4, wherein the method enriches for photoreceptor cells.

6. The method of claim 5, wherein the photoreceptor cells are cone photoreceptor cells.

7. The method of any one of claims 1-6, wherein the method enriches for photoreceptor precursor cells.

8. The method of claim 7, wherein the photoreceptor precursor cells are cone photoreceptor precursor cells.

299. The method of any one of claims 1-8, wherein the mixed population of cells comprises differentiated human iPS or embryonic stem (ES) cells.

10. The method of any one of claims 1-9, wherein the mixed population of cells comprises human retinal organoid cells.

11. The method of any one of claims 1-10, wherein the separating step is performed using fluorescence-activated cell sorting (FACS), magnetic-activated cell sorting (MACS), and / or cell panning.

12. The method of any one of claims 1-11, wherein the enriched population of cells comprises at least about 80%, at least about 85%, or at least about 90% photoreceptor and / or photoreceptor precursor cells.

13. A method of enriching photoreceptor cells and / or photoreceptor precursor cells in a mixed population of cells, the method comprising: contacting the mixed population of cells with a CD26 binding agent and, optionally, a JAM-B binding agent, allowing the photoreceptor cells and / or photoreceptor precursor cells to be bound by the CD26 binding agent and the JAM-B binding agent, if present, and separating at least a portion of the bound photoreceptor cells and / or photoreceptor precursor cells from the mixed population of cells, thereby obtaining a population of cells enriched for photoreceptor cells and / or photoreceptor precursor cells, wherein the mixed population is enriched using only the CD26 binding agent and, optionally the JAM-B binding agent, and wherein the method does not comprise contacting the mixed population of cells or the enriched population of cells with a CD29 binding agent or a CD15-SSEA1 binding agent.

14. The method of claim 13, wherein the CD26 binding agent and / or the JAM-B binding agent is an antibody or a nanobody.

15. The method of claim 13 or 14, wherein the method enriches for photoreceptor cells.

16. The method of claim 15, wherein the photoreceptor cells are cone photoreceptor cells.

17. The method of any one of claims 13-16, wherein the method enriches for photoreceptor precursor cells.3018. The method of claim 17, wherein the photoreceptor precursor cells are cone photoreceptor precursor cells.

19. The method of any one of claims 13-18, wherein the mixed population of cells comprises differentiated human iPS or ES cells.

20. The method of any one of claims 13-19, wherein the mixed population of cells comprises human retinal organoid cells.

21. The method of any one of claims 13-20, wherein the separating step is performed using FACS, MACS, and / or cell panning.

22. The method of any one of claims 13-21, wherein the enriched population of cells comprises at least about 80%, at least about 85%, or at least about 90% photoreceptor and / or photoreceptor precursor cells.

23. A method of enriching photoreceptor cells and / or photoreceptor precursor cells in a mixed population of cells, the method comprising: contacting the mixed population of cells with a CD133 binding agent and, optionally, a JAM-B binding agent, allowing the photoreceptor cells and / or photoreceptor precursor cells to be bound by the CD 133 and JAM-B binding agent, if present, and separating at least a portion of the bound photoreceptor cells and / or photoreceptor precursor cells from the mixed population of cells, thereby obtaining a population of cells enriched for photoreceptor cells and / or photoreceptor precursor cells, wherein the mixed population is enriched using only the CD133 binding agent and optionally the JAM-B binding agent, and wherein the method does not comprise contacting the mixed population of cells or the enriched population of cells with a CD29 binding agent or a CD15-SSEA1 binding agent.

24. The method of claim 23, wherein the CD133 binding agent and / or the JAM-B binding agent is an antibody or a nanobody.

25. The method of claim 23 or 24, wherein the method enriches for photoreceptor cells.

26. The method of claim 25, wherein the photoreceptor cells are cone photoreceptor cells.

27. The method of any one of claims 23-26, wherein the method enriches for photoreceptor precursor cells.

28. The method of claim 27, wherein the photoreceptor precursor cells are cone photoreceptor precursor cells.

29. The method of any one of claims 23-28, wherein the mixed population of cells comprises differentiated human iPS or ES cells.

30. The method of any one of claims 23-29, wherein the mixed population of cells comprises human retinal organoid cells.

31. The method of any one of claims 23-30, wherein the separating step is performed using FACS, MACS, and / or cell panning.

32. The method of any one of claims 23-31, wherein the enriched population of cells comprises at least about 80%, at least about 85%, or at least about 90% photoreceptor and / or photoreceptor precursor cells.

33. A population of cells obtained from differentiated iPS or embryonic stem cells or retinal organoid cells wherein at least 80%, at least 85%, at least 90%, or at least 95% of the population of cells are photoreceptor and / or photoreceptor precursor cells.

34. The population of cells of claim 33, wherein the photoreceptor and / or photoreceptor precursor cells express JAM-B on their surface.

35. The population of cells of claim 33 or 34, wherein the population of cells was obtained using the method of any one of claims 1-22.

36. A method of treating a retinal disorder, the method comprising, administering the population of cells of any one of claims 33-35.

37. The method of claim 36, wherein the method comprises administering transvitreally or via a suprachoroidal route.

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