Photoreceptor cells for retinal and macular repair

Abstract

Degenerative macular disease is the leading cause of incurable blindness. The present disclosure relates to cell-based therapeutics for retinal and macular repair, comprising defined retinal cell subtypes including short-wavelength (S), mid-wavelength (M) and long-wavelength (L) sensitive cone photoreceptors, methods for the manufacture of such cell-based therapeutics, and methods of using such therapeutics. In certain embodiments, the present disclosure is directed to a cellular composition comprising a population of L cones and M cones, wherein the ratio of L cones to M cones (L:M) falls within a predetermined range.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 63 / 235,380, filed August 20, 2021, the contents of which are incorporated by reference in their entirety and to which priority is claimed.

[0002] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH This invention was made with Government support under Grant EY030872 awarded by the National Institute of Health. The Government has certain rights in this invention.

[0003] 1. Field of the invention The present disclosure relates to cell-based therapeutics for retinal and macular repair, including defined retinal cell subtypes including rod photoreceptors, short-wavelength (S), medium-wavelength (M) and long-wavelength (L) sensitive cone photoreceptors, and associated retinal interneurons, methods for the manufacture of such cell-based therapeutics, and methods of using such therapeutics. [Background technology]

[0004] 2.Background Degenerative macular diseases are the leading cause of incurable blindness. Examples include age-related macular degeneration (AMD), Stargardt's disease, cone dystrophy, color blindness, Best's disease, mitochondrial macular degeneration, pattern dystrophy, RDS-related macular degeneration, and other forms of hereditary macular dystrophies. The macula allows high-acuity vision due to its high density of cone photoreceptors. Furthermore, the ratio of rods, S-cones, M-cones, and L-cones in the normal macula is an important determinant of normal high-acuity vision. The primary cellular cause of pathology in degenerative macular diseases is the degeneration of cone photoreceptor cells in the human macula, which results in the loss of high acuity, central vision, and photopic vision required for face recognition and reading. Summary of the Invention [Means for solving the problem]

[0005] Currently, the options for patients diagnosed with degenerative macular disease are limited.Moreover, these options typically reduce the rate of degeneration, rather than providing a way to actually repair affected tissue.Therefore, there is still a need in the art for additional therapeutic agents to address degenerative macular disease, particularly therapeutic agents that can provide repair of affected tissue.

[0006] 3. Overview In certain embodiments, the present disclosure is directed to cell-based therapeutics, e.g., cellular compositions, methods for the manufacture of such cell-based therapeutics, and methods of using such therapeutics, for retinal and macular repair comprising defined retinal cell subtypes including rod photoreceptors, short-wavelength (S), mid-wavelength (M) and long-wavelength (L) sensitive cone photoreceptors, and associated retinal interneurons.

[0007] In certain embodiments, the present disclosure is directed to a cellular composition comprising a population of L and M cones, wherein the ratio of L to M cones (L:M) falls within a predetermined range. In certain embodiments, the predetermined L:M range is about 1.3:1 to about 2.8:1. In certain embodiments, the predetermined L:M ratio is about 2:1. In certain embodiments, the population of cones comprises, as a percentage of total cones, up to about 2% S cones. In certain embodiments, the population of cones comprises, as a percentage of total cones, up to about 5% S cones.

[0008] In certain embodiments, the ratio of the populations of S, M, and L cones (S:M:L) falls within a predetermined range. In certain embodiments, S:M:L is about 1:33:66. In certain embodiments, S:M:L is about 3:33:64. In certain embodiments, the predetermined S:M:L ratio is a ratio that occurs naturally in trichromatic patients with normal color vision at a certain retinal eccentricity. In certain embodiments, the certain retinal eccentricity is at or around the foveal umbo. In certain embodiments, the inner boundary of the certain retinal eccentricity is at about 0 mm linear eccentricity and the outer boundary is at about 0.1 mm linear eccentricity. In certain embodiments, the certain retinal eccentricity is at or around the foveal center. In one particular embodiment, the inner boundary of a particular retinal eccentricity is at about 0.1 mm linear eccentricity and the outer boundary is at about 0.175 mm linear eccentricity.

[0009] In certain embodiments, the composition comprises rods ("R"), and the S:M:L:R ratio falls within a predetermined range. In certain embodiments, the predetermined S:M:L:R ratio is a ratio that occurs naturally in trichromatic patients with normal color vision at a particular retinal eccentricity. In certain embodiments, the particular retinal eccentricity is at or around the foveal center. In certain embodiments, the inner boundary of the particular retinal eccentricity is at about 0.175 mm linear eccentricity, and the outer boundary is at about 0.750 mm linear eccentricity. In certain embodiments, the cone population comprises about 10% to about 20% S cones as a percentage of total cones. In certain embodiments, the rod population comprises about 55% to about 80% rods as a percentage of total cones and rods combined. In certain embodiments, the S:M:L:R ratio is about 6:11:23:60. In certain embodiments, the particular retinal eccentricity is at or around the parafovea. In certain embodiments, the inner boundary of the particular retinal eccentricity is at about 0.750 mm linear eccentricity and the outer boundary is at about 1.50 mm linear eccentricity. In certain embodiments, the cone population comprises about 7% to about 10% S cones as a percentage of total cones. In certain embodiments, the rod population comprises about 60% to about 95% rods as a percentage of total cones and rods combined. In certain embodiments, the ratio of S:M:L:R is about 2:6:12:80. In certain embodiments, the particular retinal eccentricity is at or around the perifovea. In certain embodiments, the inner boundary of the particular retinal eccentricity is at about 1.50 mm linear eccentricity and the outer boundary is at about 3.0 mm linear eccentricity. In certain embodiments, the cone population comprises, as a percentage of total cones, about 6% to about 9% S-cones. In certain embodiments, the rod population comprises, as a percentage of total cones and rods combined, about 75% to about 95% rods. In certain embodiments, the ratio of S:M:L:R is about 1:3:7:90. In certain embodiments, the particular retinal eccentricity is at or around the peripheral macula. In certain embodiments, the inner boundary of the particular retinal eccentricity is at about 3.0 mm linear eccentricity and the outer boundary is at about 4.5 mm linear eccentricity.In certain embodiments, the rod population comprises about 55% to about 85% rods as a percentage of total cones and rods combined. In certain embodiments, the ratio of S:M:L:R is about 2:9:19:70. In certain embodiments, the particular retinal eccentricity is at or around the pericentric retina. In certain embodiments, the inner boundary of the particular retinal eccentricity is at about 4.50 mm linear eccentricity and the outer boundary is at about 6.0 mm linear eccentricity. In certain embodiments, the rod population comprises about 50% to about 80% rods as a percentage of total cones and rods combined. In certain embodiments, the ratio of S:M:L:R is about 2:11:22:65. In certain embodiments, the particular retinal eccentricity is at or around the peripheral retina. In certain embodiments, the inner boundary of the particular retinal eccentricity is at about 6.0 mm linear eccentricity and the outer boundary is at about 7.5 mm linear eccentricity. In certain embodiments, the ratio of S:M:L:R is about 3:10:22:65. In certain embodiments, the particular retinal eccentricity is at or around the far peripheral retina. In certain embodiments, the inner boundary of the particular retinal eccentricity is at about 7.50 mm linear eccentricity and the outer boundary is greater than about 7.50 mm linear eccentricity. In certain embodiments, the rod population comprises about 60% to about 90% rods as a percentage of total cones and rods combined. In certain embodiments, the ratio of S:M:L:R is about 2:7:14:75.

[0010] In certain embodiments, the present disclosure is directed to a composition comprising two regions, each region comprising a different M:L ratio. In certain embodiments, the composition comprises two regions, each region comprising a different S:M:L ratio. In certain embodiments, the composition comprises two regions, each region comprising a different S:M:L:R ratio.

[0011] In certain embodiments, the present disclosure is directed to a cellular composition comprising a population of cones, wherein one or more of the cone outer segments exhibit capacitance, and wherein the composition is characterized by a membrane current in the range of 500-2500 pA.

[0012] In certain embodiments, the present disclosure is directed to a method for preparing a cellular composition comprising a predetermined population of S, M and L cones, comprising culturing organoids such that the organoids achieve a desired ratio of S, M and L cones.

[0013] In certain embodiments, the present disclosure is directed to a method for preparing a cellular composition comprising a predetermined population of S, M and L cones, comprising: a) culturing two or more independent organoids; and b) combining cells obtained from two or more independent organoids to achieve a desired ratio of S, M and L cones.

[0014] In certain embodiments, the present disclosure is directed to a method of treating age-related macular degeneration comprising engraftment of the cellular compositions described herein into the macula of a patient in need thereof.

[0015] In certain embodiments, the present disclosure is directed to a method of treating retinal degeneration comprising engraftment of the cellular compositions described herein into the macula of a patient in need thereof.

[0016] In certain embodiments, the retinal degeneration treated by the methods of the present disclosure is due to age-related macular degeneration, Stargardt's disease, cone dystrophy, color blindness, Best's disease, mitochondrial macular degeneration, pattern dystrophy, or RDS-related macular degeneration. [Brief description of the drawings]

[0017] 4. Brief description of the drawings [Figure 1]FIG. 1 illustrates the different anatomical zones of the macula and retina that can be used to inform macular repair cell designs to regenerate the approximate cellular composition found in the zones in normal (trichromatic) subjects.

[0018] [Diagram 2] FIG. 2 provides macular repair cell designs for specific macular and retinal zones.

[0019] [Diagram 3] FIG. 3 provides specification ranges for particular macular repair cell designs.

[0020] [Figure 4] FIG. 4 shows the temporal variation in the ratio of photoreceptor identities in exemplary cultures lacking retinoic acid (RA). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0021] 5. Detailed Description The present disclosure relates to cell-based therapeutics for retinal and macular repair, including defined retinal cell subtypes including rod and short-wavelength (S), mid-wavelength (M) and long-wavelength (L) sensitive cone photoreceptors, methods for the manufacture of such cell-based therapeutics, and methods of use of such therapeutics. The cell-based therapeutics of the present disclosure are referred to herein as macular repair cells (MARCs).

[0022] In one aspect, the subject matter of the present disclosure is directed to MARC therapeutic compositions.For example, but not limited to, the present disclosure is directed to MARC compositions that include a population of cone cells, in which the ratio of S ("blue") cone, M ("green") cone and L ("red") cone falls within a certain range.As shown in FIG. 1, design variations in MARC are created so that regenerative substrates can be designed to target degenerative locations at different anterior-posterior eccentricities in the retina.The MARC variations that target each target location are designed to mimic the naturally occurring short-wavelength (S), middle-wavelength (M), long-wavelength (L) sensitive cone photoreceptor and rod (R) photoreceptor ratio (S:M:L:R ratio) in trichromatic patients with normal color vision at that eccentricity.

[0023] In another aspect, the present disclosure is directed to a method for the manufacture of MARC therapeutics.In certain embodiments, MARC therapeutics can be generated through human retinal organoid culture.For example, but not limited to, MARC therapeutics can be generated using a unique stem cell-based human retinal organoid protocol that specifically enriches for S-cones, M-cones and / or L-cones.By enriching for a specific ratio of S-cones, M-cones and / or L-cones, the manufacturing method described herein can produce MARC therapeutics that are designed to reproduce the approximate cellular composition found in (or across) a specific zone in normal three-color discrimination subjects.

[0024] In another aspect, the subject matter of the present disclosure is directed to macular regeneration therapy by MARC transplantation as a treatment to reverse functional deficits in people with retinal degenerative diseases.For example, but not limited to, retinal degenerative diseases that can be reversed by administration of the MARC composition of the present disclosure include age-related macular degeneration, Stargardt's disease, cone dystrophy, color vision deficiency, Best's disease, mitochondrial macular degeneration, pattern dystrophy, RDS-related macular degeneration and other forms of inherited macular dystrophy.As described herein, MARC therapeutics can be implanted into the macula, and such implantation can result in the regeneration of cone photoreceptor cells.Not only can MARC transplantation preserve and / or improve vision in people suffering from macular diseases, but MARC delivery is performed to ensure optimal maturation and integration of MARC therapeutics into the macula, as described in detail herein.

[0025] For clarity, and not by way of limitation, the detailed description of the presently disclosed subject matter is divided into the following subsections: 5.1 Definition 5.2 Design variations in macular repair cell composition 5.3 Methods of Producing Macular Repair Cell Compositions 5.4 Methods of Treatment Using Macular Repair Cell Compositions

[0026] 5.1.Definition The terms used herein generally have their ordinary meaning in the art, within the context of this disclosure, and in the specific context in which each term is used. Certain terms are discussed below or elsewhere in this specification to provide the practitioner with further guidance in describing the compositions and methods of the present disclosure and how to make and use them.

[0027] As used herein, the use of the words "a" or "an" when used in conjunction with the term "comprising" in the claims and / or specification can mean "one," but is also consistent with the meaning of "one or more," "at least one," and "one or more than one."

[0028] The terms "comprise(s)", "include(s)", "having", "has", "can", "contain(s)" and variations thereof, as used herein, are intended to be open-ended transitional phrases, terms or words that do not exclude the possibility of additional acts or structures. The present disclosure also contemplates other embodiments that "comprise", "consist of" and "consist essentially of" the embodiments or elements set forth herein, whether or not expressly stated.

[0029] The term "about" or "approximately" means within an acceptable error range for a particular value as determined by a person skilled in the art, which depends in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, "about" can mean within 3 standard deviations or more than 3 standard deviations, according to the practice in the art. Alternatively, "about" can mean within a range of up to 20%, preferably up to 10%, more preferably up to 5%, and more preferably still up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, preferably within 5-fold, more preferably within 2-fold of a value.

[0030] 5.2. Design Variations in Macular Repair Cell Composition In one aspect, the subject matter of the present disclosure is directed to a MARC therapeutic composition.For example, but not limited to, the present disclosure is directed to a MARC composition comprising a population of cone cells, wherein the ratio of S, M and L cones falls within a certain range.As shown in Figure 1, design variations in MARC are created so that regenerative substrates can be designed to target degenerative locations at different anterior-posterior eccentricities in the retina.The MARC variations that target each target location are designed to mimic the naturally occurring S:M:L:R ratio in trichromats with normal color vision at that eccentricity.

[0031] In certain embodiments, the MARC composition of the present disclosure is a "MARC1" composition. The MARC1 composition is designed for central foveal regeneration at or around the foveal pit at its inner border at about 0 mm linear eccentricity and its outer border at up to about 0.1 mm linear eccentricity. In certain MARC1 compositions, the L:M ratio ranges from about 1.3:1 to about 2.8:1. In certain embodiments, the L:M ratio of MARC1 is about 2:1. In certain embodiments, the MARC1 composition contains up to about 2% S cones (as a percentage of total cones). In certain embodiments, the MARC1 composition does not contain rods. In certain embodiments, the MARC1 composition has an S:M:L:R ratio that is about 1:33:66:0.

[0032] In certain embodiments, the MARC composition of the present disclosure is a "MARC2" composition. The MARC2 composition is designed for central foveal regeneration at or around the foveola at its inner border at about 0.1 mm linear eccentricity and its outer border at up to about 0.175 mm linear eccentricity. In certain MARC2 compositions, the L:M ratio ranges from about 1.3:1 to about 2.8:1. In certain embodiments, the L:M ratio of MARC2 is about 2:1. In certain embodiments, the MARC2 composition contains up to about 5% S cones (as a percentage of total cones). In certain embodiments, the MARC2 composition does not contain rods. In certain embodiments, the MARC2 composition has an S:M:L:R ratio that is about 3:33:64:0.

[0033] In certain embodiments, the MARC composition of the present disclosure is a "MARC3" composition. The MARC3 composition is designed for central regeneration at or around the fovea, with the inner border at about 0.175 mm linear eccentricity and the outer border at up to about 0.750 mm linear eccentricity. In certain MARC3 compositions, the L:M ratio ranges from about 1.3:1 to about 2.8:1. In certain embodiments, the L:M ratio of MARC3 is about 2:1. In certain embodiments, the MARC3 composition comprises about 10% to about 20% S cones (as a percentage of total cones). In certain embodiments, the MARC3 composition comprises about 55% to about 80% rods (as a percentage of total cones + rods). In certain embodiments, the MARC3 composition has an S:M:L:R ratio that is about 6:11:23:60.

[0034] In certain embodiments, the MARC composition of the present disclosure is a "MARC4" composition. The MARC4 composition is designed for regeneration at or around the parafovea at the inner border at about 0.750 mm linear eccentricity and the outer border at up to about 1.5 mm linear eccentricity. In certain MARC4 compositions, the L:M ratio ranges from about 1.3:1 to about 2.8:1. In certain embodiments, the L:M ratio of MARC4 is about 2:1. In certain embodiments, the MARC4 composition comprises about 7% to about 10% S cones (as a percentage of total cones). In certain embodiments, the MARC4 composition comprises about 60% to about 95% rods (as a percentage of total cones + rods). In certain embodiments, the MARC4 composition has an S:M:L:R ratio that is about 2:6:12:80.

[0035] In certain embodiments, the MARC composition of the present disclosure is a "MARC5" composition. The MARC5 composition is designed for regeneration at or around the perifovea at its medial border at about 1.5 mm linear eccentricity and its lateral border at up to about 3.0 mm linear eccentricity. In certain MARC5 compositions, the L:M ratio ranges from about 1.3:1 to about 2.8:1. In certain embodiments, the L:M ratio of MARC5 is about 2:1. In certain embodiments, the MARC5 composition comprises about 6% to about 9% S cones (as a percentage of total cones). In certain embodiments, the MARC5 composition comprises about 75% to about 95% rods (as a percentage of total cones + rods). In certain embodiments, the MARC5 composition has an S:M:L:R ratio that is about 1:3:7:90.

[0036] In certain embodiments, the MARC composition of the present disclosure is a "MARC6" composition. The MARC6 composition is designed for regeneration at or around the peripheral macula at an inner border at about 3.0 mm linear eccentricity and an outer border at up to about 4.5 mm linear eccentricity. In certain MARC6 compositions, the L:M ratio ranges from about 1.3:1 to about 2.8:1. In certain embodiments, the L:M ratio of MARC6 is about 2:1. In certain embodiments, the MARC6 composition comprises about 6% to about 9% S cones (as a percentage of total cones). In certain embodiments, the MARC6 composition comprises about 55% to about 85% rods (as a percentage of total cones + rods). In certain embodiments, the MARC6 composition has an S:M:L:R ratio that is about 2:9:19:70.

[0037] In certain embodiments, the MARC composition of the present disclosure is a "MARC7" composition. The MARC7 composition is designed for regeneration at or around the central-peripheral retina at an inner border at about 4.5 mm linear eccentricity and an outer border at up to about 6.0 mm linear eccentricity. In certain MARC7 compositions, the L:M ratio ranges from about 1.3:1 to about 2.8:1. In certain embodiments, the L:M ratio of MARC7 is about 2:1. In certain embodiments, the MARC7 composition comprises about 6% to about 9% S-cones (as a percentage of total cones). In certain embodiments, the MARC7 composition comprises about 50% to about 80% rods (as a percentage of total cones + rods). In certain embodiments, the MARC7 composition has an S:M:L:R ratio that is about 2:11:22:65.

[0038] In certain embodiments, the MARC composition of the present disclosure is a "MARC8" composition. The MARC8 composition is designed for regeneration at or around the peripheral retina at an inner border at about 6.0 mm linear eccentricity and an outer border at up to about 7.5 mm linear eccentricity. In certain MARC8 compositions, the L:M ratio ranges from about 1.3:1 to about 2.8:1. In certain embodiments, the L:M ratio of MARC8 is about 2:1. In certain embodiments, the MARC8 composition comprises about 7% to about 10% S cones (as a percentage of total cones). In certain embodiments, the MARC8 composition comprises about 50% to about 80% rods (as a percentage of total cones + rods). In certain embodiments, the MARC8 composition has an S:M:L:R ratio that is about 3:10:22:65.

[0039] In certain embodiments, the MARC composition of the present disclosure is a "MARC9" composition. The MARC9 composition is designed for regeneration at or around the far peripheral retina at linear eccentricities of about 7.5 mm or higher. In certain MARC9 compositions, the L:M ratio ranges from about 1.3:1 to about 2.8:1. In certain embodiments, the L:M ratio of MARC9 is about 2:1. In certain embodiments, the MARC9 composition comprises about 7% to about 10% S cones (as a percentage of total cones). In certain embodiments, the MARC9 composition comprises about 60% to about 90% rods (as a percentage of total cones + rods). In certain embodiments, the MARC9 composition has an S:M:L:R ratio that is about 2:7:14:75.

[0040] In certain embodiments, MARC designs are combined to span one or more adjacent or non-adjacent zones. For example, but not by way of limitation, the present disclosure is directed to a MARC composition comprising a population of cone cells, where the ratio of S, M and L cones falls within a particular range in one region of the composition, and the ratio of S, M and L cones falls within another particular range in another region of the composition. Such design variations in MARC can be created so that regenerative substrates can be designed to target degenerative locations that span one or more adjacent or non-adjacent anterior-posterior eccentricities. MARC variations targeting each target location can be designed to mimic the naturally occurring S:M:L:R ratio in trichromats with normal color vision at the targeted eccentricity.

[0041] In certain embodiments, the MARC composition of the present disclosure may include 1, 2, 3, 4, 5, 6, 7, 8, or 9 different regions, each of which may be designed to mimic the naturally occurring S:M:L:R ratio in trichromats with normal color vision at the targeted eccentricity. For example, without limitation, the MARC composition may include a region including a MARC1 composition and a region including a MARC2 composition. In certain embodiments, the MARC composition may include a region including a MARC2 composition and a region including a MARC3 composition. In certain embodiments, the MARC composition may include a region including a MARC3 composition and a region including a MARC4 composition. In certain embodiments, the MARC composition may include a region including a MARC4 composition and a region including a MARC5 composition. In certain embodiments, the MARC composition may include a region including a MARC5 composition and a region including a MARC6 composition. In certain embodiments, the MARC composition may include a region including a MARC6 composition and a region including a MARC7 composition. In certain embodiments, the MARC composition may include a region including a MARC7 composition and a region including a MARC8 composition. In certain embodiments, a MARC composition may include a region that includes a MARC8 composition and a region that includes a MARC9 composition.

[0042] In certain embodiments, the MARC composition may include a region including a MARC1 composition, a region including a MARC2 composition, a region including a MARC3 composition, a region including a MARC4 composition, a region including a MARC5 composition, a region including a MARC6 composition, a region including a MARC7 composition, a region including a MARC8 composition, and / or a region including a MARC9 composition. For example, and not by way of limitation, the MARC composition may include a region including a MARC1 composition, a region including a MARC2 composition, and / or a region including a MARC3 composition. In certain embodiments, the MARC composition may include a region including a MARC1 composition, a region including a MARC2 composition, a region including a MARC3 composition, and / or a region including a MARC4 composition. In certain embodiments, the MARC composition may include a region including a MARC1 composition, a region including a MARC2 composition, a region including a MARC3 composition, a region including a MARC4 composition, and / or a region including a MARC5 composition. In certain embodiments, the MARC composition may include a region including a MARC1 composition, a region including a MARC2 composition, a region including a MARC3 composition, a region including a MARC4 composition, a region including a MARC5 composition, and / or a region including a MARC6 composition. In certain embodiments, the MARC composition may include a region including a MARC1 composition, a region including a MARC2 composition, a region including a MARC3 composition, a region including a MARC4 composition, a region including a MARC5, a region including a MARC6 composition, and / or a region including a MARC7 composition. In certain embodiments, the MARC composition may include a region including a MARC1 composition, a region including a MARC2 composition, a region including a MARC3 composition, a region including a MARC4 composition, a region including a MARC5, a region including a MARC6 composition, a region including a MARC7 composition, and / or a region including a MARC8 composition. In certain embodiments, the MARC composition may include a region comprising a MARC1 composition, a region comprising a MARC2 composition, a region comprising a MARC3 composition, a region comprising a MARC4 composition, a region comprising a MARC5, a region comprising a MARC6, a region comprising a MARC7, a region comprising a MARC8 composition, and / or a region comprising a MARC9 composition.

[0043] In certain embodiments, a MARC may include discontinuous regions, such as a region including a MARC1 composition and a region including a MARC3 composition. Further non-limiting examples of such MARCs including discontinuous regions include: a MARC including a region including a MARC1 composition and a region including a MARC3, MARC4, MARC5, MARC6, MARC7, MARC8, or MARC9 composition; a MARC including a region including a MARC2 composition and a region including a MARC4, MARC5, MARC6, MARC7, MARC8, or MARC9 composition; a MARC including a region including a MARC3 composition and a region including a MARC1, MARC5, MARC6, MARC7, MARC8, or MARC9 composition; a MARC including a region including a MARC4 composition and a region including a MARC1, MARC2, MARC6, MARC7, MARC8, or MARC9 composition; a MARC including a region including a MARC5 composition and a region including a MARC 1, a MARC including a region including a MARC2, MARC3, MARC7, MARC8 or MARC9 composition; a MARC including a region including a MARC6 composition and a region including a MARC1, MARC2, MARC3, MARC4, MARC8 or MARC9 composition; a MARC including a region including a MARC7 composition and a region including a MARC1, MARC2, MARC3, MARC4, MARC5 or MARC9 composition; a MARC including a region including a MARC8 composition and a region including a MARC1, MARC1, MARC2, MARC3, MARC4, MARC5 or MARC6 composition; and a MARC including a region including a MARC9 composition and a region including a MARC1, MARC2, MARC3, MARC4, MARC5, MARC6 or MARC7 composition.

[0044] In certain embodiments, a MARC composition of the present disclosure may include a population of cone cells, where one or more of the cone outer segments exhibit large capacitance. As used herein, a MARC composition where one or more of the cone outer segments exhibit large capacitance refers to a MARC composition characterized by a membrane current in the range of 500-2500 pA. 5.3 Methods of Producing Macular Repair Cell Compositions

[0045] In one aspect, the present disclosure relates to the manufacture of MARC therapeutics.In certain embodiments, MARC can be generated through human retinal organoid culture.For example, but not limited to, MARC can be generated using a unique stem cell-based human retinal organoid protocol that specifically enriches for S-cones, M-cones and / or L-cones.By enriching for a specific ratio of S-cones, M-cones and / or L-cones, the manufacturing method described herein can produce MARC designed to reproduce the approximate cellular composition found in (or across) a specific zone in normal three-color discrimination subjects.

[0046] For example, but not limited to, the present disclosure is directed to a method for inducing the directed differentiation of cells into a population of macular repair cells by contacting cells with one or more signaling molecules under conditions that can direct the differentiation of cells into a population of macular repair cells.In certain embodiments, the cells that are induced to differentiate into a population of macular repair cells are embryonic stem cells, induced non-embryonic pluripotent cells or engineered pluripotent cells.In certain embodiments, the signaling molecules that are used to contact the cells that are induced to differentiate into a population of macular repair cells are thyroid hormone, retinoic acid, and combinations thereof.

[0047] In certain embodiments, the population of macular repair cells is prepared from organoid.For example, but not limited to, the organoid used in the preparation of the population of macular repair cells can be prepared according to the following "general organoid differentiation protocol".As the basis for the preparation and differentiation of organoid, those skilled in the art can select suitable embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs), for example, but not limited to, H7 WA07, H7iCas9 ESCs or EP1.1 iPSCs.To aggregate, the cells can then be passaged in Accutase (SCR005, Sigma) for, for example, about 12 minutes at 37°C to ensure complete dissociation. The cells can then be seeded in 96-well ultra-low attachment round-bottom Lipidure-coated plates (51011610, NOF) or ultra-low attachment microplates (7007, Corning) at 3,000 cells / well in 50 μL of mTeSR1, although alternative densities and containers are considered within the scope of this protocol. The cells can then be placed in low oxygen conditions (e.g., about 10% CO2 and about 5% O2) for about 24 hours to enhance survival. The cells naturally aggregate by gravity over the course of 24 hours.

[0048] On about day 1, the cells may be transferred to normoxic conditions (e.g., about 5% CO2). On or about days 1-3, about 50 μL of BE6.2 medium or other suitable medium containing about 3 μM Wnt inhibitor (IWR1e:681669, EMD Millipore) and about 1% (v / v) Matrigel may be added to each well. On or about days 4-9, about 100 μL of medium may be removed from each well and about 100 μL of medium may be added. On or about days 4-5, BE6.2 medium or other suitable medium containing about 3 μM Wnt inhibitor and about 1% Matrigel may be added. On or about days 6-7, BE6.2 medium or other suitable medium containing about 1% Matrigel (354230, BD Biosciences) may be added. On or about day 8-9, BE6.2 medium or other appropriate medium containing about 1% Matrigel and about 100 nM Smoothened agonist (SAG:566660, EMD Millipore) may be added.

[0049] On or about day 10, aggregates may be transferred to a 15 mL tube (or other acceptable container), rinsed about three times in about 5 mL of DMEM (11885084, Gibco) or other appropriate medium, and resuspended in BE6.2 or other appropriate medium containing about 100 nM SAG in an untreated 10 cm polystyrene Petri dish or other appropriate container. From this point on, medium may be changed about every other day. Aggregates may be monitored and manually separated if stuck together or to the bottom of the plate.

[0050] At or about days 13-16, LTR medium or other suitable medium containing about 100 nM SAG may be added. At or about days 16, retinal vesicles may be manually excised, for example, using a sharp tungsten needle. After excision, the cells may be transferred into a 15 mL tube or other acceptable container and washed about twice with about 5 mL of DMEM or other acceptable medium. At or about days 16-20, the cells may be maintained in LTR or other acceptable medium and washed about twice with about 5 mL of DMEM or other acceptable medium, and then transferred to a new plate or other acceptable container to wash away dead cells. To increase survival and differentiation, about 1.04 μM all-trans retinoic acid (ATRA; R2625; Sigma) may be added to the LTR medium or other acceptable medium at about days 20-43. As noted below, additional time windows of exposure to about 1.04 μM all-trans retinoic acid may be added depending on the desired L-cone or M-cone cell composition. About 10 μM of gamma-secretase inhibitor (DAPT, 565770, EMD Millipore) may be added to LTR or other acceptable medium at about days 28-42. In certain embodiments, organoids may be grown at low density (about 10 to about 20 per 10 cm dish) to reduce aggregation. Periodically, organoids may be thinned from the plate based on the absence of clear laminar structures indicative of proper retinal organoid growth.

[0051] In certain embodiments, the population of macular repair cells is enriched for S (blue) cones.For example, but not limited to, the population of macular repair cells enriched for S (blue) cones can be produced by the general organoid differentiation protocol described above, and the ESCs or iPSCs used to generate organoids contain Thrβ (Thrβ1 and Thrβ2) knockout.For example, but not limited to, CRISPR / Cas9 can be used, for example, in human ESCs, to delete the shared exon that codes for part of the DNA binding domain of Thrβ, as described in detail in Eldred et al.However, various other strategies can be used to knock out Thrβ expression, and are considered to be within the scope of the subject matter of the present disclosure.

[0052] In certain embodiments, the population of macular repair cells is enriched for M (green) cones.For example, but not by way of limitation, the population of macular repair cells enriched for M (green) cones can be produced by the general organoid differentiation protocol described above, but the organoid is further exposed to RA on about day 43 to about day 130, which produces a population of cells enriched for M (green) cones by about day 200.

[0053] In certain embodiments, the population of macular repair cells is enriched for L (red) cones.For example, but not limited to, the population of macular repair cells enriched for L (red) cones can be produced by the general organoid differentiation protocol described above, but the organoid is further exposed to RA from about day 130 to about day 200, and produces a population of cells enriched for L cones at day 200.

[0054] In certain embodiments, a population of macular repair cells described herein is prepared as described herein, further combined with an appropriate number of rod cells to achieve a desired S:M:L:R ratio, reproducing the approximate cellular composition found in a particular zone (or across a particular zone) in a normal trichromatic subject through a mixture of an appropriate number of cone cells combined to achieve a desired ratio of S, M, and L cones.

[0055] In certain embodiments, populations of macular repair cells can be produced, for example, in separate organoids and then mixed to achieve a desired ratio of S, M, and L cones, which are then further combined with an appropriate number of rod cells to achieve a desired S:M:L:R ratio. For example, and not by way of limitation, Table 1 provides exemplary combinations of cell compositions, including the thyroid hormone T3 and retinoic acid concentration conditions used in their production, as well as the duration of culture, for producing the MARC1-MARC9 compositions described herein. Table 1. [Table 1]

[0056] Further combinations to produce compositions that span one or more adjacent or non-adjacent zones can be produced in a similar manner, for example, by producing a cellular composition having characteristics of a first zone and pairing it with cells having characteristics of a second (or subsequent) zone.

[0057] In certain embodiments, non-neural retinal cells are depleted from the population of macular repair cells described herein.For example, but not limited to, such population of macular repair cells can be depleted from forebrain-like cells, forebrain precursor cells and / or retinal pigment epithelial cells.The markers of forebrain-like cells and / or forebrain precursor cells that can be used to verify depletion include, but are not limited to, one or more of NKX2.2, RGCC, NEUROD1, BTG2, GADD45A and GADD45G.The markers of retinal pigment epithelial cells that can be used to verify depletion include, but are not limited to, one or more of BEST1, TIMP3, GRAMD3 and PITPNA.

[0058] 5.4. METHODS OF TREATMENT USING MACULAR REPAIR CELL COMPOSITIONS In another aspect, the subject matter of the present disclosure is directed to macular regeneration therapy by MARC transplantation as a treatment to reverse functional deficits in people with retinal degenerative diseases.For example, but not limited to, retinal degenerative diseases that can be reversed by administration of the MARC composition of the present disclosure include age-related macular degeneration, Stargardt's disease, cone dystrophy, color vision deficiency, Best's disease, mitochondrial macular degeneration, pattern dystrophy, RDS-related macular degeneration and other forms of inherited macular dystrophy.As described herein, MARC therapeutics can be implanted into the macula, and such implantation can result in the regeneration of cone photoreceptor cells.Not only can MARC transplantation preserve and / or improve vision in people suffering from macular diseases, but MARC delivery is performed to ensure optimal maturation and integration of MARC therapeutics into the macula, as described in detail herein.

[0059] In certain embodiments, the methods of the present disclosure are directed to treating age-related macular degeneration, Stargardt's disease, cone dystrophies, color vision deficiencies, Best's disease, mitochondrial macular degeneration, pattern dystrophies, RDS-related macular degeneration and other forms of inherited macular dystrophies comprising engraftment of a macular repair cell composition comprising a population of cone cells into the macula of a patient in need thereof, where the ratio of S, M and L cones falls within a range of ratios corresponding to each targeted location, thereby mimicking the S:M:L:R ratio naturally occurring in trichromatic patients with normal color vision at the targeted eccentricity.

[0060] In certain embodiments, the disclosed method comprises the engraftment of a MARC1 composition in the macula of a patient in need thereof. In certain embodiments, the method comprises the engraftment of a MARC1 composition in the macula of a patient in need thereof designed for central foveal regeneration at or around the foveal pit at an inner border at about 0 mm linear eccentricity and an outer border at up to about 0.1 mm linear eccentricity. In certain embodiments, the method comprises the engraftment of a MARC1 composition in the macula of a patient in need thereof having an L:M ratio ranging from about 1.3:1 to about 2.8:1. In certain embodiments, the L:M ratio of MARC1 is about 2:1. In certain embodiments, the method comprises the engraftment of a MARC1 composition comprising up to about 2% S-cones (as a percentage of total cones) in the macula of a patient in need thereof. In certain embodiments, the method comprises the engraftment of a rod-free MARC1 composition in the macula of a patient in need thereof. In certain embodiments, the methods include engraftment of a MARC1 composition having an S:M:L:R ratio that is about 1:33:66:0 into the macula of a patient in need thereof.

[0061] In certain embodiments, the disclosed method comprises the engraftment of a MARC2 composition in the macula of a patient in need thereof. In certain embodiments, the method comprises the engraftment of a MARC2 composition in the macula of a patient in need thereof designed for central foveal regeneration at or around the fovea at its inner border at about 0.1 mm linear eccentricity and its outer border at up to about 0.175 mm linear eccentricity. In certain embodiments, the method comprises the engraftment of a MARC2 composition in the macula of a patient in need thereof having an L:M ratio ranging from about 1.3:1 to about 2.8:1. In certain embodiments, the L:M ratio of MARC2 is about 2:1. In certain embodiments, the method comprises the engraftment of a MARC2 composition comprising up to about 5% S-cones (as a percentage of total cones) in the macula of a patient in need thereof. In certain embodiments, the method comprises the engraftment of a rod-free MARC2 composition in the macula of a patient in need thereof. In certain embodiments, the methods include engraftment of a MARC2 composition having an S:M:L:R ratio that is about 3:33:64:0 into the macula of a patient in need thereof.

[0062] In certain embodiments, the disclosed method comprises the engraftment of a MARC3 composition in the macula of a patient in need thereof. In certain embodiments, the method comprises the engraftment of a MARC3 composition in the macula of a patient in need thereof, the MARC3 composition designed for central foveal regeneration at or around the fovea at an inner border at about 0.175 mm linear eccentricity and an outer border at up to about 0.750 mm linear eccentricity. In certain embodiments, the method comprises the engraftment of a MARC3 composition in the macula of a patient in need thereof, the MARC3 composition having an L:M ratio ranging from about 1.3:1 to about 2.8:1. In certain embodiments, the L:M ratio of the MARC3 is about 2:1. In certain embodiments, the method comprises the engraftment of a MARC3 composition comprising about 10% to about 20% S-cones (as a percentage of total cones) in the macula of a patient in need thereof. In certain embodiments, the method comprises engraftment in the macula of a patient in need thereof of a MARC3 composition comprising about 55% to about 80% rods (as a percentage of total cones + rods).In certain embodiments, the method comprises engraftment in the macula of a patient in need thereof of a MARC3 composition having an S:M:L:R ratio that is about 6:11:23:60.

[0063] In certain embodiments, the disclosed method comprises the engraftment of a MARC4 composition in the macula of a patient in need thereof. In certain embodiments, the method comprises the engraftment of a MARC4 composition in the macula of a patient in need thereof, the MARC4 composition designed for regeneration at or around the parafovea at an inner border at about 0.750 mm linear eccentricity and an outer border at up to about 1.5 mm linear eccentricity. In certain embodiments, the method comprises the engraftment of a MARC4 composition in the macula of a patient in need thereof, the MARC4 composition having an L:M ratio ranging from about 1.3:1 to about 2.8:1. In certain embodiments, the L:M ratio of the MARC4 is about 2:1. In certain embodiments, the method comprises the engraftment of a MARC4 composition comprising about 7% to about 10% S-cones (as a percentage of total cones) in the macula of a patient in need thereof. In certain embodiments, the method comprises engraftment in the macula of a patient in need thereof of a MARC4 composition comprising about 60% to about 95% rods (as a percentage of total cones + rods).In certain embodiments, the method comprises engraftment in the macula of a patient in need thereof of a MARC4 composition having an S:M:L:R ratio that is about 2:6:12:80.

[0064] In certain embodiments, the disclosed method comprises the engraftment of a MARC5 composition in the macula of a patient in need thereof. In certain embodiments, the method comprises the engraftment of a MARC5 composition in the macula of a patient in need thereof, the MARC5 composition designed for regeneration at or around the perifovea at an inner border at about 1.5 mm linear eccentricity and an outer border at up to about 3.0 mm linear eccentricity. In certain embodiments, the method comprises the engraftment of a MARC5 composition in the macula of a patient in need thereof, the MARC5 composition having an L:M ratio ranging from about 1.3:1 to about 2.8:1. In certain embodiments, the L:M ratio of the MARC5 is about 2:1. In certain embodiments, the method comprises the engraftment of a MARC5 composition comprising about 6% to about 9% S-cones (as a percentage of total cones) in the macula of a patient in need thereof. In certain embodiments, the method comprises engraftment in the macula of a patient in need thereof of a MARC5 composition comprising about 75% to about 95% rods (as a percentage of total cones + rods).In certain embodiments, the method comprises engraftment in the macula of a patient in need thereof of a MARC5 composition having an S:M:L:R ratio that is about 1:3:7:90.

[0065] In certain embodiments, the disclosed methods include engraftment of a MARC6 composition into the macula of a patient in need thereof. In certain embodiments, the methods include engraftment of a MARC6 composition into the macula of a patient in need thereof, the MARC6 composition being designed for regeneration at or around the peripheral macula at an inner border at about 3.0 mm linear eccentricity and an outer border at up to about 4.5 mm linear eccentricity. In certain embodiments, the methods include engraftment of a MARC6 composition into the macula of a patient in need thereof, the MARC6 composition having an L:M ratio ranging from about 1.3:1 to about 2.8:1. In certain embodiments, the L:M ratio of the MARC6 is about 2:1. In certain embodiments, the methods include engraftment of a MARC6 composition comprising about 6% to about 9% S-cones (as a percentage of total cones) into the macula of a patient in need thereof. In certain embodiments, the method comprises engraftment in the macula of a patient in need thereof of a MARC6 composition comprising about 55% to about 85% rods (as a percentage of total cones + rods).In certain embodiments, the method comprises engraftment in the macula of a patient in need thereof of a MARC6 composition having an S:M:L:R ratio that is about 2:9:19:70.

[0066] In certain embodiments, the disclosed method comprises the engraftment of a MARC7 composition in the macula of a patient in need thereof. In certain embodiments, the method comprises the engraftment of a MARC7 composition in the macula of a patient in need thereof, the MARC7 composition designed for regeneration at or around the central-peripheral retina at an inner border at about 4.5 mm linear eccentricity and an outer border at up to about 6.0 mm linear eccentricity. In certain embodiments, the method comprises the engraftment of a MARC7 composition in the macula of a patient in need thereof, the MARC7 composition having an L:M ratio ranging from about 1.3:1 to about 2.8:1. In certain embodiments, the L:M ratio of the MARC7 is about 2:1. In certain embodiments, the method comprises the engraftment of a MARC7 composition comprising about 6% to about 9% S-cones (as a percentage of total cones) in the macula of a patient in need thereof. In certain embodiments, the method comprises engraftment in the macula of a patient in need thereof of a MARC7 composition comprising about 50% to about 80% rods (as a percentage of total cones + rods).In certain embodiments, the method comprises engraftment in the macula of a patient in need thereof of a MARC7 composition having an S:M:L:R ratio that is about 2:11:22:65.

[0067] In certain embodiments, the methods of the disclosure include engraftment of a MARC8 composition into the macula of a patient in need thereof. In certain embodiments, the methods include engraftment of a MARC8 composition into the macula of a patient in need thereof, the MARC8 composition being designed for regeneration at or around the peripheral retina at an inner border at about 6.0 mm linear eccentricity and an outer border at up to about 7.5 mm linear eccentricity. In certain embodiments, the methods include engraftment of a MARC8 composition into the macula of a patient in need thereof, the MARC8 composition having an L:M ratio ranging from about 1.3:1 to about 2.8:1. In certain embodiments, the L:M ratio of the MARC8 is about 2:1. In certain embodiments, the methods include engraftment of a MARC8 composition comprising about 7% to about 10% S-cones (as a percentage of total cones) into the macula of a patient in need thereof. In certain embodiments, the method involves engraftment in the macula of a patient in need thereof of a MARC8 composition comprising about 50% to about 80% rods (as a percentage of total cones + rods).In certain embodiments, the method involves engraftment in the macula of a patient in need thereof of a MARC8 composition having an S:M:L:R ratio that is about 3:10:22:65.

[0068] In certain embodiments, the disclosed methods include engraftment of a MARC9 composition in the macula of a patient in need thereof. In certain embodiments, the methods include engraftment of a MARC9 composition designed for regeneration at or around the far peripheral retina at linear eccentricity of about 7.5 mm or higher in the macula of a patient in need thereof. In certain embodiments, the methods include engraftment of a MARC9 composition having an L:M ratio ranging from about 1.3:1 to about 2.8:1 in the macula of a patient in need thereof. In certain embodiments, the L:M ratio of the MARC9 is about 2:1. In certain embodiments, the methods include engraftment of a MARC9 composition comprising about 7% to about 10% S-cones (as a percentage of total cones) in the macula of a patient in need thereof. In certain embodiments, the methods include engraftment of a MARC9 composition comprising about 60% to about 90% rods (as a percentage of total cones + rods) in the macula of a patient in need thereof. In certain embodiments, the methods include engraftment of a MARC9 composition having an S:M:L:R ratio that is about 2:7:14:75 into the macula of a patient in need thereof.

[0069] In certain embodiments, the method includes implantation of a MARC designed to span one or more adjacent or non-adjacent zones in the macula of a patient in need thereof. For example, but not by way of limitation, the present disclosure is directed to a method including implantation of a MARC composition comprising a population of cone cells in the macula of a patient in need thereof, where the ratio of S, M and L cones falls within a particular range in one region of the composition, and the ratio of S, M and L cones falls within another particular range in another region of the composition. Such design variations in the MARC can be created so that the regenerative substrate can be designed to target degenerative locations that span one or more adjacent or non-adjacent anterior-posterior eccentricities. The MARC variations targeting each target location can be designed to mimic the naturally occurring S:M:L:R ratio in trichromatic patients with normal color vision at the targeted eccentricity.

[0070] In certain embodiments, the method includes implantation in the macula of a patient in need thereof of a MARC composition comprising 1, 2, 3, 4, 5, 6, 7, 8, or 9 distinct regions, each region may be designed to mimic the naturally occurring S:M:L:R ratio in trichromatic patients with normal color vision at a targeted eccentricity. For example, without limitation, a MARC composition for use in the methods described herein may comprise a region comprising a MARC1 composition and a region comprising a MARC2 composition. In certain embodiments, a MARC composition for use in the methods described herein may comprise a region comprising a MARC2 composition and a region comprising a MARC3 composition. In certain embodiments, a MARC composition for use in the methods described herein may comprise a region comprising a MARC3 composition and a region comprising a MARC4 composition. In certain embodiments, a MARC composition for use in the methods described herein may comprise a region comprising a MARC4 composition and a region comprising a MARC5 composition. In certain embodiments, a MARC composition for use in the methods described herein may comprise a region comprising a MARC5 composition and a region comprising a MARC6 composition. In certain embodiments, a MARC composition for use in the methods described herein may include a region that includes a MARC6 composition and a region that includes a MARC7 composition. In certain embodiments, a MARC composition for use in the methods described herein may include a region that includes a MARC7 composition and a region that includes a MARC8 composition. In certain embodiments, a MARC composition for use in the methods described herein may include a region that includes a MARC8 composition and a region that includes a MARC9 composition.

[0071] In certain embodiments, a MARC composition for use in the methods described herein may include a region comprising a MARC1 composition, a region comprising a MARC2 composition, a region comprising a MARC3 composition, a region comprising a MARC4 composition, a region comprising a MARC5 composition, a region comprising a MARC6 composition, a region comprising a MARC7 composition, a region comprising a MARC8 composition, and / or a region comprising a MARC9 composition. For example, and not by way of limitation, a MARC composition for use in the methods described herein may include a region comprising a MARC1 composition, a region comprising a MARC2 composition, and / or a region comprising a MARC3 composition. In certain embodiments, a MARC composition for use in the methods described herein may include a region comprising a MARC1 composition, a region comprising a MARC2 composition, a region comprising a MARC3 composition, and / or a region comprising a MARC4 composition. In certain embodiments, a MARC composition for use in the methods described herein may include a region comprising a MARC1 composition, a region comprising a MARC2 composition, a region comprising a MARC3 composition, and / or a region comprising a MARC4 composition. In certain embodiments, a MARC composition for use in the methods described herein may include a region comprising a MARC1 composition, a region comprising a MARC2 composition, a region comprising a MARC3 composition, a region comprising a MARC4 composition, a region comprising a MARC5 composition, and / or a region comprising a MARC6 composition. In certain embodiments, a MARC composition for use in the methods described herein may include a region comprising a MARC1 composition, a region comprising a MARC2 composition, a region comprising a MARC3 composition, a region comprising a MARC4 composition, a region comprising a MARC5, a region comprising a MARC6 composition, and / or a region comprising a MARC7 composition. In certain embodiments, a MARC composition for use in the methods described herein may include a region comprising a MARC1 composition, a region comprising a MARC2 composition, a region comprising a MARC3 composition, a region comprising a MARC4 composition, a region comprising a MARC5, a region comprising a MARC6 composition, a region comprising a MARC7 composition, and / or a region comprising a MARC8 composition.In certain embodiments, a MARC composition for use in the methods described herein may include a region comprising a MARC1 composition, a region comprising a MARC2 composition, a region comprising a MARC3 composition, a region comprising a MARC4 composition, a region comprising a MARC5, a region comprising a MARC6, a region comprising a MARC7, a region comprising a MARC8 composition, and / or a region comprising a MARC9 composition.

[0072] In certain embodiments, a MARC composition for use in the methods described herein may include discontinuous regions, such as a region including a MARC1 composition and a region including a MARC3 composition. Further non-limiting examples of such MARC compositions for use in the methods described herein that include discontinuous regions include: a MARC including a region including a MARC1 composition and a region including a MARC3, MARC4, MARC5, MARC6, MARC7, MARC8, or MARC9 composition; a MARC including a region including a MARC2 composition and a region including a MARC4, MARC5, MARC6, MARC7, MARC8, or MARC9 composition; a MARC including a region including a MARC3 composition and a region including a MARC1, MARC5, MARC6, MARC7, MARC8, or MARC9 composition; a MARC including a region including a MARC4 composition and a region including a MARC1, MARC2, MARC6, MARC7, MARC8, or MARC9 composition; a MARC including a region including a MARC4 composition and a region including a MARC1, MARC2, MARC6, MARC7, MARC8, or MARC9 composition; a MARC including a region including a MARC5 composition. MARC including a region including a MARC6 composition and a region including a MARC1, MARC2, MARC3, MARC4, MARC8 or MARC9 composition; MARC including a region including a MARC7 composition and a region including a MARC1, MARC2, MARC3, MARC4, MARC5 or MARC9 composition; MARC including a region including a MARC8 composition and a region including a MARC1, MARC1, MARC2, MARC3, MARC4, MARC5 or MARC6 composition; and MARC including a region including a MARC9 composition and a region including a MARC1, MARC2, MARC3, MARC4, MARC5, MARC6 or MARC7 composition.

[0073] In certain embodiments, the MARC delivery associated with the methods described herein is carried out using a device for accessing and delivering cells or other materials to the subretinal space.For example, but not limited to, the device for MARC delivery associated with the methods described herein may be the device described in PCT application PCT / US2019 / 045074 (WO2020028892), the entirety of which is incorporated herein by reference.

[0074] Briefly, devices for MARC delivery related to the methods described herein, such as those described in PCT Application PCT / US2019 / 045074 (WO2020028892), can access the subretinal space via the transscleral side. This is an ab extemo approach. In certain embodiments, the cell delivery device can include two stacked layers surrounded by a flexible outer surface, as shown, for example, in Figures 1-8 of PCT Application PCT / US2019 / 045074 (WO2020028892). In certain embodiments, the device can be configured to be flexible so as to conform to the natural curvature of the eye as it is advanced into the subretinal space. In certain embodiments, after the device is in place, the flexible outer surface is configured to protect the delicate tissues of the retina and retinal pigment epithelium and choroid, but there is also an easy passageway for the delivered material or cells between the two stacked layers.

[0075] In certain embodiments, the device for use in connection with the methods described herein may include an optical coherence tomography sensor directly integrated into the guide needle to allow visualization of the subretinal space during the process of opening the subretinal space. In certain embodiments, the device for use in connection with the methods described herein may include a flexible cannula and injector system to safely navigate the propagation tunnel. In certain embodiments, the device for use in connection with the methods described herein may include a plunger system that exerts force on the MARC composition, reducing or eliminating the risk of damaging it. EXAMPLES

[0076] The following examples are merely illustrative of the subject matter of the present disclosure and should not be construed as limiting in any way.

[0077] Example 1 Preparation of organoids enriched for L / M cones versus S cones As described in detail in Eldred et al., Science 2018, 362:6411 (2018):eaau6348, (Eldred et al.), which is incorporated herein by reference in its entirety, human organoids can recapitulate the cone subtype specification observed in the human retina, including the temporal generation of S cones followed by L and M cones. Furthermore, this regulation is controlled by thyroid hormone signaling, which is necessary and sufficient to control cone subtype fate through the nuclear hormone receptor, thyroid hormone receptor beta (Thrβ). As described in Eldred et al. and outlined below, this example provides an exemplary method for preparing organoids enriched for L / M cones relative to S cones.

[0078] To determine whether cone cell specification in organoids recapitulates development in the human retina, Eldred et al. compared the cone subtype signatures in human organoids with those of adult retinal tissue. Adult human retina and organoids at day 200 of differentiation showed similar ratios of S to L / M cones as indicated by expression of S-opsin or L / M-opsin (adult, S=13%, L / M=87%; organoid, S=29%, L / M=71%) (Eldred et al., Figure 1, B and C, and Figure S1A). Eldred et al. indicate that the difference in ratio is likely due to the immaturity of the organoids at approximately 6 months compared to the terminally differentiated adult retina. Eldred et al. also examined L / M cones using an antibody that recognizes both L-opsin and M-opsin proteins due to their extremely high similarity. Both S and L / M cones express the cone-rod-homeobox transcription factor (CRX), a transcription factor important for photoreceptor differentiation (Eldred et al., Figure 2, A and E), indicating proper fate specification in the organoids. Furthermore, Eldred et al. showed that cones in the organoids and retina exhibited similar morphology, with L / M cones having longer outer segments and wider inner segments than those of S cones (Eldred et al., Figure 2, B-D and F-H). Cone outer segments were also shown to be shorter in the organoids than in the adult retina, consistent with postnatal maturation (Eldred et al., Figure 2, D and H). Thus, Eldred et al. noted that cone subtypes in human retinal organoids exhibited distributions, gene expression patterns, and morphology similar to those of cones in the human retina.

[0079] Eldred et al. also examined the developmental dynamics of cone subtype specification in organoids. In the human retina, S cones are generated between embryonic weeks 11 and 34 (77–238 days), whereas L / M cones are specified later, between embryonic weeks 14 and 37 (98–259 days). Eldred et al. followed the ratio and density of S and L / M cones in organoids by antibody staining over 360 days of differentiation. Cones expressing S-opsin were first observed at day 150 (Eldred et al., Figure 2, I, L, and M). The density of S cones plateaued at day 170 (Eldred et al., Figure 2, M), a time point at which cones expressing L / M-opsin began to be observed (Eldred et al., Figure 2, J–M). The population of L / M cones increased dramatically until day 300, when they reached a steady-state density (Eldred et al., Figure 2, K–M). The 20-day difference between the onset of S-opsin and L / M-opsin expression in retinal organoids is similar to the 20-day difference observed in the appearance of S and L / M cones in the fetal retina. These observations indicate a temporal switch from S to L / M cone specification during retinal development.

[0080] Eldred et al. next performed RNA sequencing (RNA-seq) throughout 250 days of induced pluripotent stem cell (iPSC)-derived organoid development. They found that S-opsin RNA was first expressed at day 111 and plateaued at day 160, whereas L / M-opsin RNA was expressed at day 160 and remained constant after day 180, consistent with the timeline of photoreceptor maturation in organoids and fetal retina (Eldred et al., Figure 2N and Figure S1B). Furthermore, CRX RNA and CRX protein were expressed before opsin in organoids, which is similar to human development (Eldred et al., Figure 2N and Figure S1, B-G). Thus, human organoids recapitulate many aspects of the cone subtype-specific developmental timeline observed in the human retina and provide a model system for discovering mechanisms of these developmental changes.

[0081] To directly test the role of Thrβ2 in human cone subtype specification, Eldred et al. used CRISPR / Cas9 in human embryonic stem cells (ESCs) to generate a homozygous mutation resulting in premature transcription termination in the first exon of Thrβ2 (Figure S2A in Eldred et al.). Strikingly, organoids derived from these mutant stem cells showed no difference in cone subtype ratios from genotypically wild-type organoids [wild-type, S=62%, L / M=38%; Thrβ2 knockout (KO), S=59%, L / M=41%; P=0.83]. The ratio of S to L / M is high for both wild-type controls and Thrβ2 KO organoids, likely due to variability in organoid differentiation. Thus, contrary to previous suggestions based on other species, Eldred et al. do not believe that Thrβ2 is essential for cone subtype specification in humans (Figure 3, A-C in Eldred et al.).

[0082] Because Thrβ2 alone is not required for human cone subtype specification, Eldred et al. investigated whether Thrβ1 and Thrβ2 are both required for cone subtype specification in humans. To completely eliminate Thrβ function (Thrβ1 and Thrβ2), Eldred et al. used CRISPR / Cas9 in human ESCs to delete a shared exon that encodes part of the DNA-binding domain of Thrβ (Figure S2A in Eldred et al.). Thrβ null mutant retinal organoids showed complete conversion of all cones to the S subtype (wild type, S = 27%, L / M = 73%; Thrβ KO, S = 100%, L / M = 0%; P < 0.0001) (Figure 3, D–E and H in Eldred et al.). In these mutants, all cones expressed S-opsin and had S-cone morphology (Figure 3, I and J in Eldred et al.). Thus, Thrβ is required to activate L / M and repress S cone fate in the human retina.

[0083] As noted in Eldred et al., Thrβ binds with high affinity to triiodothyronine (T3), a more active form of thyroid hormone, to regulate gene expression. Furthermore, depletion or addition of T3 alters the ratio of S to M cones in rodents. Because L / M cones differentiate after S cones, Eldred et al. hypothesized that T3 acts through Thrβ later in retinal development to induce L / M cone fates and suppress S cone fates. One prediction of this hypothesis is that addition of T3 earlier in development will induce L / M fates and suppress S fates. To test this model, Eldred et al. added 20 nM T3 to ESC- and iPSC-derived organoids starting at day 20 through day 50 and continued through day 200 of differentiation. Eldred et al. observed a dramatic conversion of pyramidal cells to an L / M fate (wild type, S = 27%, L / M = 73%; wild type + T3, S = 4%, L / M = 96%; P < 0.01) ( Fig. 3, F and H , and fig. S2B in Eldred et al.). Thus, early addition of T3 is sufficient to induce L / M fate and suppress S fate.

[0084] To test whether T3 specifically acts to control cone subtype specification through Thrβ, Eldred et al. differentiated Thrβ mutant organoids by early T3 addition. The Thrβ mutation completely suppressed the effect of T3 and generated organoids with only S cones (wild type + T3, S = 4%, L / M = 96%; Thrβ KO + T3, S = 100%, L / M = 0%; P < 0.0001) (Eldred et al., Figure 3, F-H). Based on these results, Eldred et al. concluded that T3 acts through Thrβ to promote L / M cone fate and suppress S cone fate.

[0085] Eldred et al. also confirmed thyroid hormone signaling-mediated regulation of L / M-opsin expression in retinoblastoma cell lines that express L / M-opsin when treated with T3 (Fig. S2, C and D, in Eldred et al.). T3-induced activation of L / M-opsin expression was suppressed upon RNA interference knockdown of Thrβ (Fig. S2, E and F, in Eldred et al.), similar to the suppression observed in human organoids.

[0086] Eldred et al. demonstrated that early T3 addition not only converted cone cells to an L / M fate in organoids, but also dramatically increased cone density (Eldred et al., Fig. 3, F and K). Moreover, T3 acts specifically to control cone density through Thrβ (Eldred et al., Fig. 3, G and K). Early T3 addition may increase cone density by advancing and extending the temporal window for L / M cone generation.

[0087] Together, these results demonstrate that T3 signals through Thrβ to promote L / M cone fates and suppress S cone fates in developing human retinal tissue.

[0088] Materials and methods described by Eldred et al.

[0089] Cell lines. H7 ESC (WA07, WiCell) and episomal derived EP1.1 iPSC lines were used for differentiation, but other suitable cell lines are known in the art, as described above in section 5.3. WERI-Rb1 retinoblastoma cells were obtained from ATCC. Cell maintenance and organoid differentiation protocols are described in the supplementary materials of Eldred et al.

[0090] CRISPR mutations. All mutations were generated in H7 ESCs. Cells were modified to express an inducible Cas9 element. Plasmids for guide RNA (gRNA) transfection were generated by using pSpCas9(BB)-P2A-Puro plasmid, which was modified from pX459_V2.0 plasmid (62988, Addgene) by replacing T2A with P2A sequence. Mutations were confirmed using polymerase chain reaction sequencing. Gene schematics of the deletions are shown in Figure S2A of Eldred et al. Detailed transfection procedures, gRNA sequences and homology arm sequences are included in the supplementary material of Eldred et al.

[0091] Immunohistochemistry. Primary antibodies were used in Eldred et al. at the following dilutions: goat anti-SW-opsin (1:200 for organoids, 1:500 for human retina) (Santa Cruz Biotechnology), rabbit anti-LW / MW-opsin (1:200 for organoids, 1:500 for human retina) (Millipore), mouse anti-CRX (1:500) (Abnova) and mouse anti-rhodopsin (1:500) (GeneTex). All secondary antibodies were Alexa Fluor conjugated (1:400) and raised in donkey (Molecular Probes). Detailed methods for fixation, microscopy and imaging of organoids, retinas and WERI-Rb1 cells are included in the supplementary material of Eldred et al.

[0092] Organoid age. Opsin expression time course. EP1 iPSC-derived organoids for time course experiments were binned into 10-day increments for analysis in Eldred et al. Organoids were binned into day 130 [actual day 129 (n=3 organoids)], day 150 [actual day 152 (n=4 organoids)], day 170 [actual day 173 (n=2 organoids)], day 200 [actual day 194-199 (n=7 organoids)], day 290 [actual day 291 (n=3 organoids)] and day 360 [actual day 361 (n=3 organoids)]. Quantification of outer segment length and inner segment width was measured in organoids at day 361 (n=3 organoids).

[0093] Opsin expression in different conditions. iCas9 H7 ESC-derived organoids for Thrb2 KO and control were analyzed at day 200 in Eldred et al. Organoids for Thrb KO, control and wild type+T3 were analyzed at two time points in Eldred et al.: two organoids were obtained for each group at day 199 and one organoid was obtained for each group at day 277. Organoids treated with T3 were obtained at time points between days 195 and 200 for different differentiations. For each treatment group and genotype, organoids were compared to control organoids grown in parallel.

[0094] RNA-seq time course. EP1 iPSC-derived organoids were analyzed at time points ranging from day 10 to day 250 of differentiation in Eldred et al. Eldred et al. acquired samples at day 10 (n=3 organoids), day 20 (n=2 organoids), day 35 (n=3 organoids), day 69 (n=3 organoids), day 111 (n=3 organoids), day 128 (n=3 organoids), day 158 (n=2 organoids), day 173 (n=3 organoids), day 181 (n=3 organoids), day 200 (n=3 organoids), and day 250 (n=3 organoids). RNA from individual organoids was extracted by using Zymo Direct-zol RNA Microprep Kit (Zymo Research) according to the manufacturer's instructions. Libraries were prepared in Eldred et al. using an Illumina TruSeq stranded mRNA kit and sequenced on an Illumina NextSeq 500 using single 200 base pair reads.

[0095] RNA-seq time course analysis. Expression levels were quantified using Kallisto (version 0.34.1) as in Eldred et al. with the following parameters: -b 100 -1 200 -s 10 -t 20-single. Gencode release 28 global annotation was used as the reference transcriptome. The transcripts per million (TPM) values ​​(Table S1 in Eldred et al.) were then used to generate graphs in Prism and heatmaps in R using ggplot2. The distribution of transcripts was plotted to identify the best low TPM cutoff (Figure S5A in Eldred et al.). The threshold was determined to be 0.7log(TPM+1) - 5TPM - and this value was used as the inflection point for the heatmap. Heatmaps for Figure S3, A-C in Eldred et al. were similarly generated by using CPM values ​​from Hoshino et al., Dev. Cell 43, 763-779.e4 (2017).

[0096] Measurements and quantification. Measurements of retinal area and cell morphology in Eldred et al. were performed by using ImageJ software. Quantification and statistics (except for RNA-seq data) in Eldred et al. were performed in GraphPad Prism with a significance cutoff of 0.01. Statistical tests are listed in the figure legends in Eldred et al., and all error bars represent SEM.

[0097] Example 2 Preparation of organoids enriched for M versus L cones As described in detail in Hadyniak et al., bioRxiv, 2021.03.30.437763 (2021) (Hadyniak et al.), which is incorporated herein by reference in its entirety, human organoids can recapitulate the cone subtype specification observed in the human retina, including the temporal generation of L and M cones. Furthermore, this regulation is controlled by retinoic acid (RA) signaling. As described in Hadyniak et al. and outlined below, this example provides an exemplary method for preparing organoids enriched for M cones relative to L cones.

[0098] As described in Hadyniak et al., RA signaling promotes M-cone fate early and suppresses L-cone fate in human retinal organoids. Differentiation of human retinal organoids involved the addition of all-trans RA (hereafter referred to as RA) from days 20 to 43 to promote early retinal patterning (Figure S5 in Hadyniak et al.). RA was not added from days 43 to 200 ("no RA"), a time window that includes the end of primitive retinal differentiation and the full duration of cell fate specification.

[0099] Hadyniak et al. were the first to use an in situ hybridization approach to test the timing of M- and L-cone generation during human retinal organoid development. At day 120, Hadyniak et al. observed very few M- and L-cones (Figs. 4A, S6A in Hadyniak et al.). Hadyniak et al. first observed notable numbers of M- and L-cones at day 140 (Figs. 4A, S6A in Hadyniak et al.). From day 140 to day 200, the organoids were enriched for L-cones (Figs. 4A-B, S6A in Hadyniak et al.). These observations suggested that human retinal organoids differentiated using this protocol lacked the developmental cues to generate a large population of M-cones before L-cones as in human fetal development.

[0100] To test whether the addition of RA induced M-cone generation in retinal organoids, Hadyniak et al. added 1.0 μM RA over different time frames and assessed M- and L-cones at day 200 (Figure 4C in Hadyniak et al.). Organoids grown in additional RA throughout development failed to differentiate and produced minimal M- or L-cones (N=6). Addition of RA early in development, from days 43 to 130, produced almost exclusively M-cone organoids at day 200 (98.35% M, 1.65% L, 0% co-expression; Figure 4D-E in Hadyniak et al.; "Early RA"). To reinforce this observation, Hadyniak et al. performed RNA-seq on ESC-derived organoids grown in "Early RA" conditions and observed high M-opsin and minimal L-opsin expression (Figure S1C in Hadyniak et al.). Hadyniak et al. also analyzed previously published RNA-seq data for iPSC-derived organoids grown in "Early RA" conditions and observed near-exclusive expression of M-opsin (Figure S1D in Hadyniak et al.). Addition of RA later in development, from days 130 to 200, resulted in L-cone-enriched organoids at day 200 similar to organoids grown without further RA (6.35% M, 92.56% L, 1.10% co-expression; Figure 4D-E in Hadyniak et al.; "Late RA"). Hadyniak et al. did not observe significant differences in overall M / L cone density at day 200 across experimental conditions (Figure S6B in Hadyniak et al.). Together, these data indicate that RA is sufficient to induce M cones and suppress L cones early in retinal organoid development.

[0101] Materials and methods described by Hadyniak et al.

[0102] Cell line maintenance. H7 ESCs (WA07, WiCell) and episomal derived EP1.1 iPSCs were used in Hadyniak et al. for retinal organoid differentiation. Stem cells were maintained in mTeSR™ (85857, Stem Cell Technologies) on 1% (v / v) Matrigel-GFR™ (354230, BD Biosciences) coated dishes in Hadyniak et al. and grown at 37°C in a HERAcell 150i or 160i 10% CO2 and 5% O2 incubator (Thermo Fisher Scientific). Cells were passaged every 4-5 days according to confluence in Hadyniak et al. as in Wahlin et al., Sci Rep 7, 766 (2017), and cells were passaged with Accutase (SCR005, Sigma) for 7-12 minutes to dissociate into single cells. Cells in Accutase were added 1:2 to mTeSR™1 plus 5 μM blebbistatin (Bleb, B0560, Sigma), pelleted at 150 g for 5 min, suspended in mTeSR™1 plus Blebbistatin, and plated at 5,000-15,000 cells per well in 6-well plates. Cells were fed with mTeSR™ 48 hours after passaging and every 24 hours until passage again. No antibiotics were used to minimize cell stress.

[0103] Weri-Rb-1 retinoblastoma cells were obtained from ATCC and maintained in Hadyniak et al. in RPMI 1640 medium (11875135, Gibco) + 10% fetal bovine serum (16140071, Gibco) + 1x penicillin-streptomycin (30-002-CI, Corning) at 37°C in a HERAcell 150i or 160i 5% CO2 incubator (Thermo Fisher Scientific). Cells were pelleted at 150g for 5 min and resuspended in fresh medium to obtain approximately 1x10 5 ~2×10 6 cells / mL were passaged every 4 days into uncoated flasks.

[0104] Cell lines were tested monthly for mycoplasma using MycoAlert (LT07, Lonza).

[0105] Cell culture media. Stem cell medium mTeSR1 (85857, StemCell Technologies). E6 supplement: 970 μg / mL insulin (11376497001, Roche), 535 μg / mL holo-transferrin (T0665, Sigma), 3.20 mg / mL L-ascorbic acid (A8960, Sigma), 0.7 μg / mL sodium selenite (S5261, Sigma). BE6.2 medium for early retinal differentiation: 2.5% E6 supplement (as above), 2% B27 supplement (50x) minus vitamin A (12587010, Gibco), 1% Glutamax (35050061, Gibco), 1% NEAA (11140050, Gibco), 1 mM pyruvate (11360070, Gibco) and 0.87 mg / mL NaCl in DMEM (11885084, Gibco). LTR (long-term retina) medium: 2% B27 supplement (50x) (17504044, Gibco) in DMEM (11885084, Gibco), 10% heat-inactivated FBS (16140071, Gibco), 25% F12 (11765062, Gibco) containing 1 mM sodium pyruvate, 1% NEAA, 1% Glutamax and 1 mM taurine (T-8691, Sigma). RPMI+ supplement medium: 10% heat-inactivated FBS (16140071, Gibco), 2.5% penicillin (30-002-CI, Corning) in RPMI medium 1640 (11875135, Gibco).

[0106] Retinoic acid treatment: for organoids, 1.04 μM all-trans retinoic acid (ATRA; R2625; Sigma) in LTR.

[0107] Thyroid hormone treatment: for Weri-Rb1 cells, 100 nM T3 (T6397, Sigma) in RPMI+ supplemented medium.

[0108] Organoid Differentiation. Organoids were differentiated from H7 WA07, H7iCas9 ESCs or EP1.1 iPSCs as described in Eldred et al. 2018 with minor variations (Figure S5 in Hadyniak et al.). Pluripotent stem cells were well maintained. Cultures with minimal to no spontaneous differentiation were used for aggregation. To aggregate, cells were passaged in Accutase (SCR005, Sigma) for 12 min at 37 °C to ensure complete dissociation. Cells were seeded in 96-well ultra-low attachment round-bottom Lipidure-coated plates (51011610, NOF) or ultra-low attachment microplates (7007, Corning) at 3,000 cells / well in 50 μL of mTeSR1. Cells were placed in hypoxic conditions (10% CO2 and 5% O2) for 24 h to enhance survival. The cells were allowed to naturally aggregate by gravity for 24 hours.

[0109] On day 1, cells were transferred to normoxic conditions (5% CO2). On days 1-3, 50 μL of BE6.2 medium containing 3 μM Wnt inhibitor (IWR1e:681669, EMD Millipore) and 1% (v / v) Matrigel was added to each well. On days 4-9, 100 μL of medium was removed from each well and 100 μL of medium was added. On days 4-5, BE6.2 medium containing 3 μM Wnt inhibitor and 1% Matrigel was added. On days 6-7, BE6.2 medium containing 1% Matrigel (354230, BD Biosciences) was added. On days 8-9, BE6.2 medium containing 1% Matrigel and 100 nM Smoothened agonist (SAG:566660, EMD Millipore) was added.

[0110] On day 10, aggregates were transferred to 15 mL tubes, rinsed three times in 5 mL DMEM (11885084, Gibco), and resuspended in BE6.2 containing 100 nM SAG in untreated 10 cm polystyrene Petri dishes. From this point on, medium was changed every other day. Aggregates were monitored and manually separated if they stuck together or to the bottom of the plate.

[0111] On days 13-16, LTR medium containing 100 nM SAG was added. On day 16, retinal vesicles were manually excised using a sharp tungsten needle. After excision, cells were transferred into a 15 mL tube and washed twice with 5 mL DMEM. On days 16-20, cells were maintained in LTR and washed twice with 5 mL DMEM before being transferred to a new plate and dead cells were washed away. To increase survival and differentiation, 1.04 μM all-trans retinoic acid (ATRA; R2625; Sigma) was added to LTR medium on days 20-43. An additional time window of 1.04 μM was added depending on the experimental conditions. 10 μM gamma-secretase inhibitor (DAPT, 565770, EMD Millipore) was added to LTR on days 28-42. Organoids were grown at low density (10-20 per 10 cm dish) to reduce clumping. Periodically, organoids were thinned from the plates based on the absence of clear, thin layer structures, which indicate proper retinal organoid growth.

[0112] RNA-Seq Experiments. EP1 iPSC-derived organoids were pre-grown and analyzed as described by Eldred et al. at time points ranging from day 10 to day 250 of differentiation. Samples were acquired at day 10 (N=3), day 20 (N=2), day 35 (N=3), day 69 (N=3), day 111 (N=3), day 128 (N=3), day 158 (N=2), day 173 (N=3), day 181 (N=3), day 200 (N=3) and day 250 (N=3).

[0113] H7 ESC-derived organoids were obtained on day 329 (N=3).

[0114] Weri-Rb-1 samples were grown for 4 days in control RPMI+supplemented medium or RPMI+supplemented medium treated with T3 (N=1).

[0115] RNA from individual samples was extracted using the Zymo Direct-zol RNA Microprep Kit (R2062, Zymo Research) according to the manufacturer's instructions. Libraries were prepared using the Illumina TruSeq stranded mRNA kit and sequenced on an Illumina NextSeq 500 using single 75 bp reads.

[0116] Preparation and cryosectioning of human retina and organoids. Human retina. Donor samples were snap frozen on dry ice at 10.9 hours postmortem and stored at -80°C. Human eyes were brought to room temperature in 1x PBS and retinas were dissected from the eyes. Retinas were fixed in 10% neutral buffered formalin (HT501128, Sigma) for 45 minutes and washed in 1x PBS. Small sections of retina were mounted in Tissue-Tek OCT compound (4583, Sakura), placed on dry ice to freeze and stored at -80°C. Retinas were sectioned at 10 μm sections. Slides were air-dried for 6 hours to overnight with a postfixation step of 15 minutes in 10% neutral buffered formalin (HT501128, Sigma) and washed in 1x PBS. Slides were dried and stored at -80°C for less than 3 months before use.

[0117] Organoids. Organoids were fixed in 10% neutral buffered formalin (HT501128, Sigma) for 45 minutes and washed in 1x PBS. Organoids were placed in 25% sucrose in 0.1M phosphate buffer overnight, then mounted in Tissue-Tek OCT compound (4583, Sakura), frozen on dry ice, and stored at -80°C. Organoids were sectioned in 10 μm sections. Slides were air-dried for 6 hours to overnight with a post-fixation step of 15 minutes in 10% neutral buffered formalin (HT501128, Sigma) and washed in 1x PBS. Slides were dried and stored at -80°C for less than 3 months before use.

[0118] Organoid cell lines used in Hadyniak et al.: No RA: H7 ESC (N=3); Early RA: H7 ESC (N=1), H7 iCas9 ESC (N=2); and Late RA: H7iCas9 ESC (N=5).

[0119] RNA in situ hybridization. BaseScope RNA in situ hybridization was performed according to the manufacturer's instructions with some modifications. Probe sequences were designed by ACD Biotechne based on the OPN1MW and OPN1LW mRNA sequences NM_000513.2 NM_020061.5 from human genome hg38.

[0120] Sections were brought to room temperature from storage at -80°C and rewetted in 1x PBS. Samples were pretreated according to the manufacturer's instructions: RNAscope hydrogen peroxide for 10 minutes, followed by one wash in dH2O, then two washes in 1x PBS.

[0121] RNAscope Protease III was applied for 15 minutes in a humid chamber at a 1:15 dilution in 1x PBS for HEK293 cells. RNAscope Protease IV was applied for 20 minutes in a humid chamber for organoids and human eye samples. Samples were washed twice with 1x PBS.

[0122] Probes were added to samples at the manufacturer's suggested concentrations in HybEZ humidity-controlled racks with lids and inserts and placed in a HybEZ oven for 2 hours at 40° C. Samples were washed twice for 2 minutes in 1× RNAscope wash buffer.

[0123] Amplification and color development washes were performed using the manufacturer's reagents with no modifications to the recommended concentrations in Hadyniak et al.. All washes were performed at room temperature (RT) or at 40°C in a HybEZ oven in a HybEZ humidity-controlled rack with lid and insert.

[0124] Two 2-minute washes in 1× RNAScope buffer were performed between each reagent wash. The final wash was in tap water. Slides were baked in a HybEZ oven at 65° C. for a minimum of 30 minutes. Samples were preserved with VectaMount (Vector Laboratories, H-5000) mounting medium and a sealed coverslip.

[0125] Imaging of organoids. All serially sectioned organoids were imaged and manually counted. Organoids with less than 150 cones (n≦150) were removed from the analysis in Hadyniak et al. Statistical tests are listed in the figure legends in Hadyniak et al., and all error bars represent SEM.

[0126] Example 3 Temporal variation in the ratio of photoreceptor species in culture conditions lacking retinoic acid (RA) Retinal organoids were grown in medium lacking exogenous RA addition after day 43 (the end of early retinal development) as generally described in Examples 1 and 2. Organoids were stained for S-opsin and M / L-opsin at various time points throughout development. From this data, the density of cone subtypes was quantified over time. As shown in Figure 4, the implementation of this protocol results in S-cones being identified first, which constitute 100% of cone cells. As M / L cone specification begins, a subpopulation of cones co-express S-opsin and M / L opsin. This co-expression is later resolved, and over time, M / L cones constitute the majority of cone subtypes. At day 130, more than 50% of cones are of M / L identity, and this approximate ratio persists at further time points studied.

[0127] The contents of all figures and all references, patents and published patent applications and accession numbers cited throughout this application are hereby expressly incorporated by reference.

Claims

1. A cellular composition comprising a population of L cones and M cones, wherein the ratio of L cones to M cones (L:M) falls within a predetermined range.

2. 2. The cellular composition of claim 1, wherein the predetermined L:M range is from about 1.3:1 to about 2.8:1, or is about 2:

1.

3. The cellular composition of claim 1, wherein the population of cones comprises up to about 2% S cones as a percentage of total cones, or up to about 5% S cones as a percentage of total cones.

4. The cellular composition of claim 1, wherein the ratio of the populations of S, M and L cones (S:M:L) falls within a predetermined range, and the S:M:L ratio is selected from the group consisting of about 1:33:66, about 3:33:64, and ratios naturally occurring in trichromatic patients with normal color vision at a particular retinal eccentricity.

5. The cellular composition of claim 4, wherein the specific retinal eccentricity is (i) at or around the foveal pit, (ii) at or around the foveal pit and the inner boundary of the specific retinal eccentricity is at a linear eccentricity of about 0 mm and the outer boundary is at a linear eccentricity of about 0.1 mm, (iii) at or around the foveal center, or (iv) at or around the foveal center and the inner boundary of the specific retinal eccentricity is at a linear eccentricity of about 0.1 mm and the outer boundary is at a linear eccentricity of about 0.175 mm.

6. The cellular composition of claim 1 , wherein the composition comprises rods ("R") and the S:M:L:R ratio falls within a predetermined range.

7. The cellular composition of claim 6, wherein the predetermined S:M:L:R ratio is a ratio that occurs naturally in a trichromatic patient with normal color vision at a particular retinal eccentricity, and the S:M:L:R ratio is selected from the group consisting of about 6:11:23:60, about 2:6:12:80, about 1:3:7:90, about 2:9:19:70, about 2:11:22:65, 3:10:22:65 and 2:7:14:

75.

8. The particular retinal eccentricity is (i) at or around the foveal center, (ii) at or around the foveal center with the inner boundary of the particular retinal eccentricity at about 0.175 mm linear eccentricity and the outer boundary at about 0.750 mm linear eccentricity, (iii) at or around the parafovea, (iv) at or around the parafovea with the inner boundary of the particular retinal eccentricity at about 0.750 mm linear eccentricity and the outer boundary at about 1.50 mm linear eccentricity, (v) at or around the perifovea, (vi) at or around the perifovea with the inner boundary of the particular retinal eccentricity at about 1.50 mm linear eccentricity and the outer boundary at about 3.0 mm linear eccentricity, (vii) at or around the peripheral macula, or (viii) at or around the peripheral macula with the inner boundary of the particular retinal eccentricity.

8. The cellular composition of claim 7, wherein the boundary is at about 3.0 mm linear eccentricity and the outer boundary is at about 4.5 mm linear eccentricity; (ix) at or around the central peripheral retina; (x) at or around the central peripheral retina, wherein the inner boundary of the particular retinal eccentricity is at about 4.50 mm linear eccentricity and the outer boundary is at about 6.0 mm linear eccentricity; (xi) at or around the peripheral retina; (xii) at or around the peripheral retina, wherein the inner boundary of the particular retinal eccentricity is at about 6.0 mm linear eccentricity and the outer boundary is at about 7.5 mm linear eccentricity; (xiii) at or around the far peripheral retina; or (xiv) at or around the far peripheral retina, wherein the inner boundary of the particular retinal eccentricity is at about 7.50 mm linear eccentricity and the outer boundary is greater than about 7.50 mm linear eccentricity.

9. The cellular composition described in claim 6, wherein the population of cones as a percentage of total cones is selected from a group comprising about 10% to about 20%, about 7% to about 10%, or about 6% to about 9% S cones.

10. The cellular composition of claim 6, wherein the population of rods as a percentage of total cones and rods combined comprises about 55% to about 80% rods, or about 60% to about 95% rods, about 75% to about 95% rods, about 55% to about 85% rods, about 50% to about 80% rods, or 60% to about 90% rods.

11. The cellular composition of claim 1, wherein the composition comprises two regions, each region comprising a different M:L ratio, or the composition comprises two regions, each region comprising a different S:M:L ratio.

12. A method for preparing a cellular composition comprising a predetermined population of S cones, M cones and L cones, the method comprising culturing the organoids so that the organoids achieve a desired ratio of S cones to M cones to L cones.

13. 1. A method for preparing a cellular composition comprising a predetermined population of S, M and L cones, comprising: a) culturing two or more independent organoids; b) combining cells from the two or more independent organoids to achieve a desired ratio of S to M to L cones. A method comprising:

14. A cellular composition described in any one of claims 1 to 11 for treating age-related macular degeneration or retinal degeneration, including engraftment into the macula of a patient in need of treatment for age-related macular degeneration or retinal degeneration.

15. The cellular composition of claim 14, wherein the retinal degeneration is caused by age-related macular degeneration, Stargardt's disease, cone dystrophy, color blindness, Best's disease, mitochondrial macular degeneration, pattern dystrophy, or RDS-associated macular degeneration.

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