Retinal pigment epithelial cell therapy

Abstract

This disclosure describes compositions of retinal pigment epithelial (RPE) cells, such as RPE cells derived from autologous induced pluripotent stem cells, and their use in the treatment of eye disorders. In particular, this disclosure provides preparations comprising retinal pigment epithelial (RPE) cells differentiated from induced pluripotent stem cells (iPSCs), wherein at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, or higher) of the RPE cells are PMEL17(+) / CD140b(+) / GD2(-) / CD184(-).

Description

Technical Field

[0001] Cross - reference to Related Applications This application claims the benefit under 35 U.S.C. § 119(e) of U.S. Provisional Patent Application No. 63 / 500,482, filed on May 5, 2023, which is hereby incorporated by reference in its entirety.

Background Art

[0002] Background Some degenerative diseases of the eye cause permanent vision loss due to retinal pigment epithelium (RPE) cell dysfunction. The treatment of these diseases represents a large unmet medical need.

Summary of the Invention

Means for Solving the Problems

[0003] Summary The present disclosure provides, inter alia, a preparation comprising retinal pigment epithelium (RPE) cells differentiated from induced pluripotent stem cells (iPSCs), wherein at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95% or higher) of the RPE cells are PMEL17(+) / CD140b(+) / GD2( - ) / CD184( - ). In some embodiments, about 80% (e.g., about 85%, 90%, 91%, 92%, 93%, 94%, 95% or higher) of the RPE cells are PMEL17(+) / CD140b(+) / GD2( - ) / CD184( - ).

[0004] In some embodiments, at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95% or higher) of the RPE cells express RPE65, BEST1, RLBP1, and MerTK. In some embodiments, about 80% (e.g., about 85%, 90%, 91%, 92%, 93%, 94%, 95% or higher) of the RPE cells express RPE65, BEST1, RLBP1, and MerTK.

[0005] In some embodiments, for example, less than 0.002% of RPE cells are positive for Lin28, Oct4, or Klf4 when detected by digital droplet PCR (ddPCR). In some embodiments, less than 0.002% of RPE cells are positive for Lin28 when detected by digital droplet PCR (ddPCR), and the RPE cells do not have measurable expression of Oct4 or Klf4.

[0006] In some embodiments, the culture of the preparation contains RPE cells in which at least 95% (e.g., at least 96%, 97%, 98%, 99%, or higher) exhibit confluent hexagonal cells, pigmentation, cobblestone structure, well-defined membrane boundaries, and / or polarization (e.g., as assessed using ezrin staining). In some embodiments, the culture of the preparation contains RPE cells in which at least 95% (e.g., at least 96%, 97%, 98%, 99%, or higher) RPE cells exhibit confluent hexagonal cells, pigmentation, cobblestone morphology, well-defined membrane boundaries, and polarization (e.g., as assessed using ezrin staining). In some embodiments, the culture of the preparation contains RPE cells in which about 95% (e.g., about 96%, 97%, 98%, 99%, or higher) RPE cells exhibit confluent hexagonal cells, pigmentation, cobblestone configuration, well-defined membrane boundaries, and / or polarization (e.g., as assessed using ezrin staining). In some embodiments, the culture of the preparation contains RPE cells in which about 95% (e.g., about 96%, 97%, 98%, 99%, or higher) RPE cells exhibit confluent hexagonal cells, pigmentation, cobblestone morphology, and well-defined membrane boundaries. In some embodiments, the culture of the preparation contains RPE cells in which approximately 95% (e.g., approximately 96%, 97%, 98%, 99%, or higher) of the RPE cells exhibit confluent hexagonal cells, pigmentation, cobblestone structure, well-defined membrane boundaries, and polarization (e.g., as assessed using ezrin staining).

[0007] In some embodiments, (a) RPE cells are cultured for about 1 to 8 weeks and then have a pressure of at least 100 ohms / cm². 2(b) RPE cells exhibit a transepithelial electrical resistance (TEER) of at least 000 ohms / cm², (c) RPE cells exhibit differential secretion of VEGF at a ratio of basal secretion to apical secretion greater than 0.5, and / or (d) at least 50% of RPE cells exhibit the ability to phagocytose photoreceptor outer segments, as measured, for example, by flow cytometry. In some embodiments, (a) RPE cells exhibit at least 100 ohms / cm² after being cultured for about 1 to 8 weeks. 2 (b) RPE cells exhibit transepithelial electrical resistance (TEER), (c) RPE cells exhibit differential secretion of VEGF in a ratio of basal secretion to apical secretion greater than 0.5, (d) RPE cells exhibit differential secretion of PEDF in a ratio of apical secretion to basal secretion greater than 0.5, and (d) at least 50% of RPE cells exhibit the ability to phagocytose photoreceptor outer segments, as measured, for example, by flow cytometry.

[0008] In some embodiments, during passage via an implantation device (e.g., a 41g device), at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, or higher) of RPE cells are viable, as determined by, for example, trypan blue staining. In some embodiments, during passage via an implantation device (e.g., a 41g device), approximately 80% (e.g., approximately 85%, 90%, 91%, 92%, 93%, 94%, 95%, or higher) of RPE cells are viable, as determined by, for example, trypan blue staining.

[0009] In some embodiments, during passage via an implantation device (e.g., a 41g device), at least 80% of RPE cells (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, or higher) reach at least approximately 50 ohms / cm² during culture, for example, 1–8 weeks. 2 , at least about 75 ohms / cm 2 at least approximately 100 ohms / cm² 2 , at least about 125 ohms / cm 2 , at least about 150 ohms / cm2 at least about 175 ohms / cm 2 or at least about 200 ohms / cm 2 of TEER. In some embodiments, upon passage through a seeding device (e.g., a 41g device), about 80% (e.g., about 85%, 90%, 91%, 92%, 93%, 94%, 95% or higher) of the RPE cells have a TEER of at least about 50 ohms / cm, at least about 75 ohms / cm, at least about 100 ohms / cm, at least about 125 ohms / cm, at least about 150 ohms / cm, at least about 175 ohms / cm, or at least about 200 ohms / cm, for example, within 1 - 8 weeks of culture. 2 at least about 75 ohms / cm 2 at least about 100 ohms / cm 2 at least about 125 ohms / cm 2 at least about 150 ohms / cm 2 at least about 175 ohms / cm 2 or at least about 200 ohms / cm 2 of TEER.

[0010] In some embodiments, when stored at room temperature for about 6 hours, at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95% or higher) of the RPE cells are viable, as determined by, for example, trypan blue staining. In some embodiments, when stored at room temperature for about 6 hours, about 80% (e.g., about 85%, 90%, 91%, 92%, 93%, 94%, 95% or higher) of the RPE cells are viable, as determined by, for example, trypan blue staining.

[0011] In some embodiments, the iPSC is derived from fibroblasts or peripheral blood mononuclear cells.

[0012] In some embodiments, the preparation contains a suspension of RPE cells. In some embodiments, the preparation contains a scaffold, matrix, or bioink, and the RPE cells are seeded, cultured, printed, or embedded on, in, or within the scaffold, matrix, or bioink.

[0013] In some embodiments, the preparation contains approximately 10,000 to 1,500,000 RPE cells.

[0014] In another embodiment, the Disclosure provides a preparation comprising retinal pigment epithelial (RPE) cells differentiated from induced pluripotent stem cells (iPSCs), the preparation having undergone at least one freeze / thaw cycle and having a viability of at least 80%, 90%, or 95% (as determined, e.g., by trypan blue staining) compared to a reference preparation comprising RPE cells differentiated from iPSCs and having undergone at least one freeze / thaw cycle. In some embodiments, the preparation has undergone at least one freeze / thaw cycle and has a viability of about 80%, 90%, or 95% (as determined, e.g., by trypan blue staining) compared to a reference preparation comprising RPE cells differentiated from iPSCs and having undergone at least one freeze / thaw cycle. In some embodiments, after at least one freeze / thaw cycle of the preparation, at least 80%, 90%, or 95% of the RPE cells of the preparation are viable (as determined, e.g., by trypan blue staining). In some embodiments, after at least one freeze / thaw cycle of the preparation, approximately 80%, 90%, or 95% of the RPE cells in the preparation were viable (as determined, e.g., by trypan blue staining). In some embodiments, after at least one freeze / thaw cycle, the preparation was cultured in culture medium for approximately 2 to 6 weeks, e.g., approximately 4 weeks.

[0015] In some embodiments, at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, or higher) of RPE cells are PMEL17(+) / CD140b(+) / GD2(-) / CD184(-). In some embodiments, approximately 80% (e.g., approximately 85%, 90%, 91%, 92%, 93%, 94%, 95%, or higher) of RPE cells are PMEL17(+) / CD140b(+) / GD2(-) / CD184(-).

[0016] In some embodiments, at least 80% of RPE cells (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, or higher) express RPE65, BEST1, RLBP1, and MerTK. In some embodiments, approximately 80% of RPE cells (e.g., approximately 85%, 90%, 91%, 92%, 93%, 94%, 95%, or higher) express RPE65, BEST1, RLBP1, and MerTK.

[0017] In some embodiments, for example, less than 0.002% of RPE cells are positive for Lin28, Oct4, or Klf4 when detected by digital droplet PCR (ddPCR). In some embodiments, less than 0.002% of RPE cells are positive for Lin28 when detected by digital droplet PCR (ddPCR), and the RPE cells do not have measurable expression of Oct4 or Klf4.

[0018] In some embodiments, the culture of the preparation contains RPE cells in which at least 95% (e.g., at least 96%, 97%, 98%, 99%, or higher) exhibit confluent hexagonal cells, pigmentation, cobblestone structure, well-defined membrane boundaries, and / or polarization (e.g., as assessed using ezrin staining). In some embodiments, the culture of the preparation contains RPE cells in which approximately 95% (e.g., approximately 96%, 97%, 98%, 99%, or higher) of the RPE cells exhibit confluent hexagonal cells, pigmentation, cobblestone structure, well-defined membrane boundaries, and / or polarization (e.g., as assessed using ezrin staining).

[0019] In some embodiments, (a) RPE cells are cultured for about 1 to 8 weeks and then have a pressure of at least 100 ohms / cm². 2(b) RPE cells exhibit a transepithelial electrical resistance (TEER) of at least 000 ohms / cm², (c) RPE cells exhibit differential secretion of VEGF at a ratio of basal secretion to apical secretion greater than 0.5, and / or (d) at least 50% of RPE cells exhibit the ability to phagocytose photoreceptor outer segments, as measured, for example, by flow cytometry. In some embodiments, (a) RPE cells exhibit at least 100 ohms / cm² after being cultured for about 1 to 8 weeks. 2 (b) RPE cells exhibit transepithelial electrical resistance (TEER), (c) RPE cells exhibit differential secretion of VEGF in a ratio of basal secretion to apical secretion greater than 0.5, (d) RPE cells exhibit differential secretion of PEDF in a ratio of apical secretion to basal secretion greater than 0.5, and (d) at least 50% of RPE cells exhibit the ability to phagocytose photoreceptor outer segments, as measured, for example, by flow cytometry.

[0020] In some embodiments, during passage via an implantation device (e.g., a 41g device), at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, or higher) of RPE cells are viable, as determined by, for example, trypan blue staining. In some embodiments, during passage via an implantation device (e.g., a 41g device), approximately 80% (e.g., approximately 85%, 90%, 91%, 92%, 93%, 94%, 95%, or higher) of RPE cells are viable, as determined by, for example, trypan blue staining.

[0021] In some embodiments, during passage via an implantation device (e.g., a 41g device), at least 80% of RPE cells (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, or higher) reach at least approximately 50 ohms / cm² during culture, for example, 1–8 weeks. 2 , at least about 75 ohms / cm 2 at least approximately 100 ohms / cm² 2 , at least about 125 ohms / cm 2 , at least about 150 ohms / cm2 At least approximately 175 ohms / cm² 2 , or at least about 200 ohms / cm² 2 This shows the TEER. In some embodiments, during passaging via an implantation device (e.g., a 41g device), approximately 80% of RPE cells (e.g., approximately 85%, 90%, 91%, 92%, 93%, 94%, 95%, or higher) reach at least approximately 50 ohms / cm² during, for example, 1–8 weeks of culture. 2 , at least about 75 ohms / cm 2 at least approximately 100 ohms / cm² 2 , at least about 125 ohms / cm 2 , at least about 150 ohms / cm 2 , at least about 175 ohms / cm 2 , or at least about 200 ohms / cm² 2 This indicates the TEER.

[0022] In some embodiments, after storage at room temperature for about 6 hours, at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, or higher) of RPE cells remain viable, as determined by, for example, trypan blue staining.

[0023] In some embodiments, iPSCs are derived from fibroblasts or peripheral blood mononuclear cells.

[0024] In some embodiments, the preparation comprises a suspension of RPE cells. In some embodiments, the preparation comprises a scaffold, matrix, or bioink, in which the RPE cells are seeded, cultured, printed, or embedded on the scaffold, matrix, or bioink.

[0025] In some embodiments, the preparation contains approximately 10,000 to 1,500,000 RPE cells.

[0026] In another embodiment, the Disclosure provides a method for producing a preparation of RPE cells, comprising: growing fibroblasts from a skin sample or peripheral blood mononuclear cells from a whole blood sample; producing iPSCs from the fibroblasts or peripheral blood mononuclear cells; differentiating the iPSCs into a culture of RPE cells; and passage the culture of RPE cells with a dissociation reagent for at least 10 minutes until at least 95% (e.g., about 95%, about 96%, about 97%, about 98%, about 99%, or more) of the cultured RPE cells exhibit confluent hexagonal cells, pigmentation, cobblestone morphology and well-defined membrane boundaries, thereby producing a preparation of RPE cells. In some embodiments, the Method further comprises subjecting the preparation to at least one freeze / thaw cycle.

[0027] In some embodiments, at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, or higher) of the RPE cells in the preparation are PMEL17(+) / CD140b(+) / GD2(-) / CD184(-). In some embodiments, approximately 80% (e.g., approximately 85%, 90%, 91%, 92%, 93%, 94%, 95%, or higher) of the RPE cells in the preparation are PMEL17(+) / CD140b(+) / GD2(-) / CD184(-).

[0028] In some embodiments, at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, or higher) of the RPE cells in the preparation express RPE65, BEST1, RLBP1, and MerTK. In some embodiments, approximately 80% (e.g., approximately 85%, 90%, 91%, 92%, 93%, 94%, 95%, or higher) of the RPE cells in the preparation express RPE65, BEST1, RLBP1, and MerTK.

[0029] In some embodiments, for example, less than 0.002% of the RPE cells in the preparation are positive for Lin28, Oct4, or Klf4 when detected by digital droplet PCR (ddPCR). In some embodiments, less than 0.002% of the RPE cells in the preparation are positive for Lin28 when detected by digital droplet PCR (ddPCR), and the RPE cells do not have measurable expression of Oct4 or Klf4.

[0030] In some embodiments, (a) RPE cells are cultured for about 1 to 8 weeks and then have a pressure of at least 100 ohms / cm². 2 (b) RPE cells exhibit a transepithelial electrical resistance (TEER) of at least 000 ohms / cm², (c) RPE cells exhibit differential secretion of VEGF at a ratio of basal secretion to apical secretion greater than 0.5, and / or (d) at least 50% of RPE cells exhibit the ability to phagocytose photoreceptor outer segments, as measured, for example, by flow cytometry. In some embodiments, (a) RPE cells exhibit at least 100 ohms / cm² after being cultured for about 1 to 8 weeks. 2 (b) RPE cells exhibit transepithelial electrical resistance (TEER), (c) RPE cells exhibit differential secretion of VEGF in a ratio of basal secretion to apical secretion greater than 0.5, (d) RPE cells exhibit differential secretion of PEDF in a ratio of apical secretion to basal secretion greater than 0.5, and (d) at least 50% of RPE cells exhibit the ability to phagocytose photoreceptor outer segments, as measured, for example, by flow cytometry.

[0031] In some embodiments, during passage via an implantation device (e.g., a 41g device), at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, or higher) of RPE cells are viable, as determined by, for example, trypan blue staining. In some embodiments, during passage via an implantation device (e.g., a 41g device), approximately 80% (e.g., approximately 85%, 90%, 91%, 92%, 93%, 94%, 95%, or higher) of RPE cells are viable, as determined by, for example, trypan blue staining.

[0032] In some embodiments, after storage at room temperature for about 6 hours, at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, or higher) of RPE cells remain viable, as determined by, for example, trypan blue staining.

[0033] In another aspect, the present disclosure provides a preparation of RPE cells prepared by any of the methods described herein.

[0034] In another embodiment, the disclosure provides a method for treating a subject suffering from or at risk of an eye disorder, comprising administering a preparation of RPE cells to the subject's eye. In some embodiments, the RPE cells are autologous to the subject. In some embodiments, the preparation is introduced into any area of ​​the subject's eye.

[0035] In some embodiments, the preparation comprises RPE cells differentiated from induced pluripotent stem cells (iPSCs), wherein at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, or higher) of the RPE cells are PMEL17(+) / CD140b(+) / GD2(-) / CD184(-). In some embodiments, approximately 80% (e.g., approximately 85%, 90%, 91%, 92%, 93%, 94%, 95%, or higher) of the RPE cells are PMEL17(+) / CD140b(+) / GD2(-) / CD184(-).

[0036] In some embodiments, at least 80% of RPE cells (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, or higher) express RPE65, BEST1, RLBP1, and MerTK. In some embodiments, approximately 80% of RPE cells (e.g., approximately 85%, 90%, 91%, 92%, 93%, 94%, 95%, or higher) express RPE65, BEST1, RLBP1, and MerTK.

[0037] In some embodiments, for example, less than 0.002% of RPE cells are positive for Lin28, Oct4, or Klf4 when detected by digital droplet PCR (ddPCR). In some embodiments, less than 0.002% of RPE cells are positive for Lin28 when detected by digital droplet PCR (ddPCR), and the RPE cells do not have measurable expression of Oct4 or Klf4.

[0038] In some embodiments, the culture of the preparation contains RPE cells in which at least 95% (e.g., at least 96%, 97%, 98%, 99%, or higher) exhibit confluent hexagonal cells, pigmentation, cobblestone structure, well-defined membrane boundaries, and / or polarization (e.g., as assessed using ezrin staining). In some embodiments, the culture of the preparation contains RPE cells in which approximately 95% (e.g., approximately 96%, 97%, 98%, 99%, or higher) of the RPE cells exhibit confluent hexagonal cells, pigmentation, cobblestone structure, well-defined membrane boundaries, and / or polarization (e.g., as assessed using ezrin staining).

[0039] In some embodiments, (a) RPE cells are cultured for about 1 to 8 weeks and then have a pressure of at least 100 ohms / cm². 2(b) RPE cells exhibit a transepithelial electrical resistance (TEER) of at least 000 ohms / cm², (c) RPE cells exhibit differential secretion of VEGF at a ratio of basal secretion to apical secretion greater than 0.5, and / or (d) at least 50% of RPE cells exhibit the ability to phagocytose photoreceptor outer segments, as measured, for example, by flow cytometry. In some embodiments, (a) RPE cells exhibit at least 100 ohms / cm² after being cultured for about 1 to 8 weeks. 2 (b) RPE cells exhibit transepithelial electrical resistance (TEER), (c) RPE cells exhibit differential secretion of VEGF in a ratio of basal secretion to apical secretion greater than 0.5, (d) RPE cells exhibit differential secretion of PEDF in a ratio of apical secretion to basal secretion greater than 0.5, and (d) at least 50% of RPE cells exhibit the ability to phagocytose photoreceptor outer segments, as measured, for example, by flow cytometry.

[0040] In some embodiments, during passage via an implantation device (e.g., a 41g device), at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, or higher) of RPE cells are viable, as determined by, for example, trypan blue staining. In some embodiments, during passage via an implantation device (e.g., a 41g device), approximately 80% (e.g., approximately 85%, 90%, 91%, 92%, 93%, 94%, 95%, or higher) of RPE cells are viable, as determined by, for example, trypan blue staining.

[0041] In some embodiments, during passage via an implantation device (e.g., a 41g device), at least 80% of RPE cells (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, or higher) reach at least approximately 50 ohms / cm² during culture, for example, 1–8 weeks. 2 , at least about 75 ohms / cm 2 at least approximately 100 ohms / cm² 2 , at least about 125 ohms / cm 2 , at least about 150 ohms / cm2 At least approximately 175 ohms / cm² 2 , or at least about 200 ohms / cm² 2 This shows the TEER. In some embodiments, during passaging via an implantation device (e.g., a 41g device), approximately 80% of RPE cells (e.g., approximately 85%, 90%, 91%, 92%, 93%, 94%, 95%, or higher) reach at least approximately 50 ohms / cm² during, for example, 1–8 weeks of culture. 2 , at least about 75 ohms / cm 2 at least approximately 100 ohms / cm² 2 , at least about 125 ohms / cm 2 , at least about 150 ohms / cm 2 , at least about 175 ohms / cm 2 , or at least about 200 ohms / cm² 2 This indicates the TEER.

[0042] In some embodiments, after storage at room temperature for about 6 hours, at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, or higher) of RPE cells remain viable, as determined by, for example, trypan blue staining.

[0043] In some embodiments, iPSCs are derived from fibroblasts or peripheral blood mononuclear cells.

[0044] In some embodiments, the preparation comprises a suspension of RPE cells. In some embodiments, the preparation comprises a scaffold, matrix, or bioink, in which the RPE cells are seeded, cultured, printed, or embedded on the scaffold, matrix, or bioink.

[0045] In some embodiments, the preparation contains approximately 10,000 to 1,500,000 RPE cells.

[0046] In another embodiment, the disclosure provides a method for treating a subject suffering from or at risk of an eye disorder, comprising administering a preparation of RPE cells to the subject's eye. In some embodiments, the RPE cells are autologous to the subject. In some embodiments, the preparation is introduced into any area of ​​the subject's eye.

[0047] In some embodiments, the preparation comprises RPE cells differentiated from induced pluripotent stem cells (iPSCs), and the preparation has undergone at least one freeze / thaw cycle and has a viability of at least 80%, 90%, or 95% (as determined, e.g., by trypan blue staining) compared to a reference preparation comprising RPE cells differentiated from iPSCs but having undergone at least one freeze / thaw cycle. In some embodiments, the preparation has undergone at least one freeze / thaw cycle and has a viability of about 80%, 90%, or 95% (as determined, e.g., by trypan blue staining) compared to a reference preparation comprising RPE cells differentiated from iPSCs but having undergone at least one freeze / thaw cycle. In some embodiments, after at least one freeze / thaw cycle of the preparation, at least 80%, 90%, or 95% of the RPE cells in the preparation are viable (as determined, e.g., by trypan blue staining). In some embodiments, after at least one freeze / thaw cycle of the preparation, approximately 80%, 90%, or 95% of the RPE cells in the preparation were viable (as determined, e.g., by trypan blue staining). In some embodiments, after at least one freeze / thaw cycle, the preparation was cultured in culture medium for approximately 2 to 6 weeks, e.g., approximately 4 weeks.

[0048] In some embodiments, at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, or higher) of RPE cells are PMEL17(+) / CD140b(+) / GD2(-) / CD184(-). In some embodiments, approximately 80% (e.g., approximately 85%, 90%, 91%, 92%, 93%, 94%, 95%, or higher) of RPE cells are PMEL17(+) / CD140b(+) / GD2(-) / CD184(-).

[0049] In some embodiments, at least 80% of RPE cells (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, or higher) express RPE65, BEST1, RLBP1, and MerTK. In some embodiments, approximately 80% of RPE cells (e.g., approximately 85%, 90%, 91%, 92%, 93%, 94%, 95%, or higher) express RPE65, BEST1, RLBP1, and MerTK.

[0050] In some embodiments, for example, less than 0.002% of RPE cells are positive for Lin28, Oct4, or Klf4 when detected by digital droplet PCR (ddPCR). In some embodiments, less than 0.002% of RPE cells are positive for Lin28 when detected by digital droplet PCR (ddPCR), and the RPE cells do not have measurable expression of Oct4 or Klf4.

[0051] In some embodiments, the culture of the preparation contains RPE cells in which at least 95% (e.g., at least 96%, 97%, 98%, 99%, or higher) exhibit confluent hexagonal cells, pigmentation, cobblestone structure, well-defined membrane boundaries, and / or polarization (e.g., as assessed using ezrin staining). In some embodiments, the culture of the preparation contains RPE cells in which approximately 95% (e.g., approximately 96%, 97%, 98%, 99%, or higher) of the RPE cells exhibit confluent hexagonal cells, pigmentation, cobblestone structure, well-defined membrane boundaries, and / or polarization (e.g., as assessed using ezrin staining).

[0052] In some embodiments, (a) RPE cells are cultured for about 1 to 8 weeks and then have a pressure of at least 100 ohms / cm². 2(b) RPE cells exhibit a transepithelial electrical resistance (TEER) of at least 000 ohms / cm², (c) RPE cells exhibit differential secretion of VEGF at a ratio of basal secretion to apical secretion greater than 0.5, and / or (d) at least 50% of RPE cells exhibit the ability to phagocytose photoreceptor outer segments, as measured, for example, by flow cytometry. In some embodiments, (a) RPE cells exhibit at least 100 ohms / cm² after being cultured for about 1 to 8 weeks. 2 (b) RPE cells exhibit transepithelial electrical resistance (TEER), (c) RPE cells exhibit differential secretion of VEGF in a ratio of basal secretion to apical secretion greater than 0.5, (d) RPE cells exhibit differential secretion of PEDF in a ratio of apical secretion to basal secretion greater than 0.5, and (d) at least 50% of RPE cells exhibit the ability to phagocytose photoreceptor outer segments, as measured, for example, by flow cytometry.

[0053] In some embodiments, during passage via an implantation device (e.g., a 41g device), at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, or higher) of RPE cells are viable, as determined by, for example, trypan blue staining. In some embodiments, during passage via an implantation device (e.g., a 41g device), approximately 80% (e.g., approximately 85%, 90%, 91%, 92%, 93%, 94%, 95%, or higher) of RPE cells are viable, as determined by, for example, trypan blue staining.

[0054] In some embodiments, during passage via an implantation device (e.g., a 41g device), at least 80% of RPE cells (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, or higher) reach at least approximately 50 ohms / cm² during culture, for example, 1–8 weeks. 2 , at least about 75 ohms / cm 2 at least approximately 100 ohms / cm² 2 , at least about 125 ohms / cm 2 , at least about 150 ohms / cm2 At least approximately 175 ohms / cm² 2 , or at least about 200 ohms / cm² 2 This shows the TEER. In some embodiments, during passaging via an implantation device (e.g., a 41g device), approximately 80% of RPE cells (e.g., approximately 85%, 90%, 91%, 92%, 93%, 94%, 95%, or higher) reach at least approximately 50 ohms / cm² during, for example, 1–8 weeks of culture. 2 , at least about 75 ohms / cm 2 at least approximately 100 ohms / cm² 2 , at least about 125 ohms / cm 2 , at least about 150 ohms / cm 2 , at least about 175 ohms / cm 2 , or at least about 200 ohms / cm² 2 This indicates the TEER.

[0055] In some embodiments, after storage at room temperature for about 6 hours, at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, or higher) of RPE cells remain viable, as determined by, for example, trypan blue staining.

[0056] In some embodiments, iPSCs are derived from fibroblasts or peripheral blood mononuclear cells.

[0057] In some embodiments, the preparation comprises a suspension of RPE cells. In some embodiments, the preparation comprises a scaffold, matrix, or bioink, in which the RPE cells are seeded, cultured, printed, or embedded on the scaffold, matrix, or bioink.

[0058] In some embodiments, the preparation contains approximately 10,000 to 1,500,000 RPE cells.

[0059] Other features, purposes, and advantages of this disclosure will become apparent in the following detailed description. However, it should be understood that the detailed description illustrates embodiments of the invention, but is given only as examples and not as a limitation. Various changes and modifications within the scope of the invention will become apparent to those skilled in the art from the detailed description.

[0060] definition A or An: The articles "a" and "an" are used herein to refer to one or more (i.e., at least one) grammatical objects of the article. For example, "element" means one or more elements.

[0061] Administration: As used herein, the term “administration” typically means the administration (e.g., a composition or treatment) to a subject or system (e.g., one or more cells, tissues, organisms, etc., or containing thereof) and means achieving the delivery of a drug that is, is contained in, or otherwise delivered or produced by such a composition or treatment.

[0062] Approximately or about: As used herein, the terms “approximately” or “about” refer to values ​​similar to the reference values ​​stated, when applied to one or more of the values ​​for which they are intended. In certain embodiments, unless otherwise specified or evident from the context (except where such numbers exceed 100% of the possible values), the terms “approximately” or “about” refer to a range of values ​​that fall within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than or less than) the reference values ​​stated.

[0063] Biodegradability: As used herein, the term “biodegradable” refers to a material that, once introduced into a cell, is broken down (by cellular mechanisms such as enzymatic degradation, hydrolysis, and / or a combination thereof) into components that allow the cell to be reused or discarded without causing significant toxicity to the cell. In certain embodiments, the components produced by the degradation of a biodegradable material are biocompatible and therefore do not induce significant inflammation and / or other adverse effects in vivo. In some embodiments, biodegradable polymer materials decompose into their component monomers. In some embodiments, the degradation of a biodegradable material (e.g., including a biodegradable polymer material) includes hydrolysis of ester bonds. Alternatively or additionally, in some embodiments, the degradation of a biodegradable material (e.g., including a biodegradable polymer material) includes cleavage of urethane bonds. Examples of biodegradable polymers include, but are not limited to, polymers of hydroxy acids such as lactic acid and glycolic acid, including, poly(hydroxyl acid), poly(lactic acid) (PLA), poly(glycolic acid) (PGA), and poly(lactic acid-co-glycolic acid) (PLGA), as well as copolymers with PEG, polyanhydrides, poly(ortho)esters, polyesters, polyurethanes, poly(butyric acid), poly(valeric acid), poly(caprolactone), poly(hydroxyalkanoates), poly(lactide-co-caprolactone), blends thereof, and copolymers. Many naturally occurring polymers are also biodegradable, for example. Examples include extracellular matrix (ECM)-derived scaffolds, proteins such as albumin, collagen, gelatin, and prolamin, such as zein, and polysaccharides such as alginates, cellulose derivatives, and polyhydroxyalkanoates, such as polyhydroxybutyrate blends and their copolymers. Those skilled in the art will be able to recognize or determine if such polymers have biocompatible and / or biodegradable derivatives (for example, related to the parent polymer by substantially identical structures differing only by the substitution or addition of certain chemical groups, as is known in the art).

[0064] Determining: Many of the methodologies described herein include a “determining” step. A person skilled in the art will understand, upon reading this specification, that such “determining” can be achieved by using, or by using, any of the various techniques available to a person skilled in the art, including, for example, certain techniques expressly referenced herein. In some embodiments, determining includes manipulating a physical sample. In some embodiments, determining includes considering and / or manipulating data or information, for example, using a computer or other processing unit adapted to perform the relevant analysis. In some embodiments, determining includes receiving relevant information and / or material from a source. In some embodiments, determining includes comparing one or more features of a sample or entity with an equivalent reference.

[0065] Differentiation: As used herein, the term “differentiation” refers to the process by which unspecialized (”unconstrained”) or less specialized cells acquire the characteristics of specialized cells, such as RPE cells. In some embodiments, differentiated cells or differentiation-inducing cells occupy a more specialized (”constrained”) position within a cell lineage. For example, iPSCs, when treated with appropriate differentiation factors in a cell culture medium, can differentiate into a variety of more differentiated cell types, such as neural or hematopoietic stem cells, lymphocytes, cardiomyocytes, and other cell types. In some embodiments, appropriate methods, differentiation factors, and cell culture media for differentiating pluripotent and multipotent cell types into more differentiated cell types are well known to those skilled in the art. In some embodiments, the term “constrained” is applied to the differentiation process to refer to cells that have progressed through the differentiation pathway to a point where, under normal circumstances, they would continue to differentiate into a particular cell type or subset of cell types, or would continue to differentiate into a less differentiated cell type, under normal circumstances.

[0066] Differentiation Markers: As used herein, the terms “differentiation marker,” “differentiation marker gene,” or “differentiation gene” refer to a gene or protein whose expression indicates cell differentiation occurring within a cell, such as a pluripotent cell. In some embodiments, differentiation marker genes include, but are not limited to, the following genes: RPE65, CRALBP, PEDF, bethroffin-1 (BEST1), RLBP1, merTK, MITF, OTX2, PAX2, PAX6, premelanosome proteins (gp-100 or PMEL, e.g., PMEL-17), CD140b, tyrosinase, and / or ZO1.

[0067] Expression: As used herein, the term “expression” of a nucleic acid sequence refers to the generation of any gene product from a nucleic acid sequence. In some embodiments, the gene product may be a transcript. In some embodiments, the gene product may be a polypeptide. In some embodiments, the expression of a nucleic acid sequence includes one or more of the following: (1) the preparation of an RNA template from a DNA sequence (e.g., by transcription), (2) the processing of the RNA transcript (e.g., by splicing, editing, etc.), (3) the translation of the RNA into a polypeptide or protein, and / or (4) post-translational modification of the polypeptide or protein.

[0068] Gene product or expression product: As used herein, the terms “gene product” or “expression product” generally refer to RNA transcribed from a gene (before and / or after processing) or polypeptides encoded by RNA transcribed from a gene (before and / or after modification).

[0069] Induced Pluripotent Stem Cells: The term “induced pluripotent stem cells” or “iPSC” as used herein refers to stem cells obtained from somatic cells (e.g., adult, neonatal, or fetal) that have been differentiated through a process called reprogramming (e.g., dedifferentiation). In some embodiments, the reprogrammed cells can differentiate into tissues of all three germ layers: mesoderm, endoderm, and ectoderm. iPSCs are not found in nature.

[0070] Marker: As used herein, a marker refers to an entity or part whose presence or level is characteristic of a particular state or event. In some embodiments, the presence or level of a particular marker may be characteristic of a particular type of cell or cell stage.

[0071] Pharmaceutical composition: As used herein, the term “pharmaceutical composition” refers to an activator formulated with one or more pharmaceutically acceptable carriers. In some embodiments, the activator is present in a unit dose amount appropriate for administration in a therapeutic regimen that exhibits a statistically significant probability of achieving a predetermined therapeutic effect when administered to the relevant population.

[0072] To prevent: As used herein in relation to disease, the terms “to prevent,” “prevent,” and “prevent” refer to the prevention of disease in mammals, such as humans, and include (a) avoiding or eliminating the disease, (b) influencing predisposition to the disease, or (c) preventing or delaying the onset of at least one symptom of the disease.

[0073] Reference: As used herein, the term “reference” refers to a standard or control on which a comparison is made. For example, in some embodiments, the active substance, animal, individual, population, sample, sequence, or value of interest is compared to the active substance, animal, individual, population, sample, sequence, or value of a reference or control. In some embodiments, the reference or control is tested and / or determined substantially concurrently with the test or determination of interest. In some embodiments, the reference or control is a historical reference or control, embodied in tangible media as necessary. Typically, as understood by those skilled in the art, the reference or control is determined or characterized under conditions or circumstances equivalent to those under evaluation. Those skilled in the art will understand that there is sufficient similarity to justify reliance on and / or comparison to a particular possible reference or control.

[0074] Reprogramming: As used herein, the terms “reprogramming” or “dedifferentiation” refer to methods of increasing the potency of cells or dedifferentiating cells to a less differentiated state. For example, in some embodiments, cells with increased potency have greater developmental plasticity (i.e., they can differentiate into more cell types) compared to the same cells that are not reprogrammed. That is, in some embodiments, reprogrammed cells are less differentiated than the same cells that are not reprogrammed. In some embodiments, “reprogramming” refers to dedifferentiating somatic cells or pluripotent stem cells into induced pluripotent stem cells, also known as iPSCs. Appropriate methods for producing iPSCs from somatic cells or pluripotent stem cells are well known to those skilled in the art.

[0075] Subject: As used herein, the term “Subject” means an organism, typically a mammal (e.g., a human, and in some embodiments, a prenatal human form). In some embodiments, the Subject suffers from a relevant disease, disorder, or condition. In some embodiments, the Subject is susceptible to a disease, disorder, or condition. In some embodiments, the Subject exhibits one or more symptoms or features of a disease, disorder, or condition. In some embodiments, the Subject exhibits no symptoms or features of a disease, disorder, or condition. In some embodiments, the Subject is a person who has one or more features characteristic of susceptibility to or risk of a disease, disorder, or condition. In some embodiments, the Subject is a patient. In some embodiments, the Subject is an individual who is and / or has been diagnosed and / or treated.

[0076] Afflicted: An individual “afflicted” with a disease, disorder, and / or condition exhibits symptoms of one or more of the diseases, disorders, and / or conditions, and / or is diagnosed with a disease, disorder, or condition.

[0077] Therapeutic Dose: As used herein, the term “therapeutic dose” means the amount of the RPE cell or compositional preparation described herein that gives a therapeutic effect to the subject being treated in a reasonable benefit / risk ratio applicable to any medical treatment. The therapeutic effect may be objective (i.e., measurable by several tests or markers) or subjective (i.e., the subject exhibits signs of or feels an effect). In particular, “therapeutic dose” means the amount of the RPE cell or compositional preparation that is effective in treating, improving or preventing a particular disease or condition, or in exhibiting a detectable therapeutic or preventive effect, e.g., improving symptoms associated with the disease, preventing or delaying the onset of the disease, and / or reducing the severity or frequency of symptoms of the disease. Therapeutic doses may be administered in a dosing regimen that may contain multiple unit doses. For any particular preparation of RPE cells or composition, the therapeutic dose (and / or appropriate unit dose in an effective dosing regimen) may vary, for example, depending on the route of administration and in combination with other pharmaceuticals. Furthermore, the specific therapeutically effective dose (and / or unit dose) for any particular subject may depend on a variety of factors, including the disorder being treated and its severity, the activity of the particular drug used, the particular composition used, the subject's age, weight, overall health, sex and diet, administration time, route of administration, and / or rate of excretion or metabolism, duration of treatment, and similar factors well known in the medical field.

[0078] Treatment: As used herein, the term “treatment” (also “treat” or “treating”) means any administration of any preparation of RPE cells or compositions described herein that partially or completely alleviates, improves, reduces, inhibits, delays the onset, reduces the severity, and / or reduces the incidence of one or more symptoms or features of a particular disease, disorder, and / or condition. Such treatment may be for subjects that do not show signs of the disease, disorder, and / or condition in question, and / or for subjects that show only initial signs of the disease, disorder, and / or condition. Alternatively or additionally, such treatment may be for subjects that show established signs of one or more of the disease, disorder, and / or condition in question. [Brief explanation of the drawing]

[0079] Brief explanation of the drawing [Figure 1] Figure 1 shows a flowchart of an exemplary AiPSC-RPE manufacturing process.

[0080] [Figure 2A] Figure 2A shows steps 1 and 2 of an exemplary morphological scoring system. [Figure 2B] Figure 2B shows steps 3 and 4 of an exemplary morphological scoring system.

[0081] [Figure 3A] Figure 3A shows the viability of AiPSC-RPE cell products retained in the final formulation over 0, 2, 4, and 6 hours compared to freshly thawed AiPSC-RPE cell products. AiPSC-RPE cell products cultured before the preparation of the final formulation had higher viability. [Figure 3B] Figure 3B shows the potency measured by TEER for AiPSC-RPE cell products that were freshly thawed, cultured for 1 week, or cultured for 4 weeks. AiPSC-RPE cell products cultured before the preparation of the final formulation had a larger ohm*cm².

[0082] [Figure 4] Figure 4 shows the viability of the AiPSC-RPE final product (AiPSC-RPE cultured before preparation in the final product) in the final formulation up to 6 hours after passing through the delivery device. The viability was over 90%. Lonza RPE was used as a positive control.

[0083] [Figure 5A] Figure 5A shows the differential secretion of the cytokine VEGF from the AiPSC-RPE end product after passing through the delivery device. [Figure 5B] Figure 5B shows the differential secretion of the cytokine PEDF from AiPSC-RPE products after passing through the delivery device. A ratio greater than 1 indicates healthy RPE cells. Lonza RPE is the positive control.

[0084] [Figure 6A-B] Figure 6A shows the percentage of AiPSC-RPE products expressing PMEL17, as measured by flow cytometry. Figure 6B shows the percentage of AiPSC-RPE products expressing CD140b, as measured by flow cytometry. [Figure 6C-D] Figure 6C shows the percentage of AiPSC-RPE products expressing GD2, as measured by flow cytometry. Figure 6D shows the percentage of AiPSC-RPE products expressing CD184, as measured by flow cytometry.

[0085] [Figure 7] Figure 7 is a schematic diagram illustrating an exemplary method for evaluating parallel cultures from a typical RPE bank.

[0086] [Figure 8]Figure 8 shows the thickness of the outer granular layer (ONL) as assessed from OCT images of RCS rats at 4, 8, and 12 weeks post-injection. Vehicle control animals were treated with BSS+ solution only, and untreated animals were not injected. AiPSC-RPE cell lines 1:01F1i1R1, AiPSC-RPE cell line 2:02F1i1R1, AiPSC-RPE cell line 3:13F1i1R1, and cell line 4:13F1i2R1. N=6 RCS rats, mean ± standard deviation.

[0087] [Figure 9] Figure 9 shows a panel of images of stained retinal frozen sections 4 weeks after transplantation with the AiPSC-RPE cell line 01F1i1R1, taken at 20x magnification. From left to right in the upper panel are STEM101 (human nucleus), RPE65 (RPE cell maturation marker), and DAPI (control nucleus), and a merged image. The lower panel shows controls with only secondary antibodies for STEM101, RPE65, RLBP1, and DAPI. The images show the persistence of AiPSC-RPE 4 weeks after transplantation.

[0088] [Figure 10] Figure 10 shows a panel of images of stained retinal frozen sections 12 weeks after transplantation with the AiPSC-RPE cell line 02F1i1R1, taken at 63x magnification. From left to right in the upper panel are RPE65, TRA-1-85 (human cell surface antigen), DAPI (nuclear staining), and merge. The lower panel shows a control with only secondary antibodies for DAPI, RPE65, and TRA-1-85. The images show the persistence of AiPSC-RPE 12 weeks after transplantation.

[0089] [Figure 11] Figure 11 shows images of stained retinal frozen sections 12 weeks after transplantation with AiPSC-RPE cell line 02F1i1R1, taken at 5x magnification. Merged images, DAPI, RPE65, TRA-1-85 (human cell surface antigen). The images show migration and integration of AiPSC-RPE cells from the injection site throughout the retina.

[0090] [Figure 12]Figure 12 shows optokinetic gain measured in RCS rats on days 24, 54, and 88. Vehicle control animals were injected with BSS+ solution only, while untreated animals were not. AiPSC-RPE cell lines: 01F1i1R1 and 02F1i1R1.

[0091] [Figure 13] Figure 13 shows the full-field ERG (39.8 cd·sec / m2) measured in RCS rats at weeks 4, 8, and 12. Vehicle control animals were injected with BSS+ solution only, while untreated animals were not. AiPSC-RPE cell lines: 01F1i1R1, 02F1i1R1, 13F1i1R1, and 13F1i2R1. [Modes for carrying out the invention]

[0092] General description of a specific embodiment This disclosure is based in part on the discovery of certain characteristics exhibited by RPE cells, such as RPE cells differentiated from iPSCs, that correlate with increased viability, purity, functionality, stability, and phenotypic effects. Certain aspects of this disclosure relate to the use of characteristics such as criteria, e.g., quality control criteria, for the preparation of RPE cells used as cell therapies for treating subjects having or suffering from ocular disorders. This disclosure is also based in part on the discovery of methods for producing RPE cell preparations that result in RPE cells exhibiting such characteristics, e.g., quality control criteria.

[0093] Several degenerative diseases of the eye cause permanent vision loss due to dysfunction of retinal pigment epithelial (RPE) cells. Treatment of these diseases represents a significant unmet medical need. Clinical trials using human embryonic stem cells (hESCs) have been described (Schwartz et al., Lancet 379(9817):713-20(2012); Schwartz et al., Lancet 385(9967):509-16(2015)), and similarly, published trials using induced pluripotent stem cell (iPSC)-derived RPE cells to treat patients with macular dystrophy have also been described (Mandai et al., N.Engl.J.Med. 376(11):1038-1046(2017)). This disclosure is based on the discovery of a novel method for generating patient-specific autologous iPSC-derived RPE cells for transplantation.

[0094] In most mammals, including humans, the photoreceptor (PR) layer plays a role in converting light into signals that can be received by the brain. While the PR layer is crucial for vision, other layers of the eye, including the rPE located between the choroid and the PR, are equally important. A healthy RPE is organized as a polarized monolayer with tight junctions and helps maintain PR function by recycling photopigments, phagocytosing the outer segments of the PR, transporting ions and small molecules between the retina and choroid, maintaining Bruch's membrane, and absorbing stray light to enable better image resolution. Therefore, the RPE layer is important for PR function and health. (Strauss, Physiol. Rev. 85(3):845-81(2005)).

[0095] Unfortunately, many diseases involve the loss of RPE and PR cells in the eye, leading to blindness. Diseases such as age-related macular degeneration (AMD), Stargardt macular dystrophy (SMD), and retinitis pigmentosa (RP) initially cause cellular dysfunction, followed by the loss of cells including RPE, ultimately leading to permanent blindness. Furthermore, other disorders such as Best's disease and macular telangiectasia can also result in the loss of RPE and PR cells (Bitner et al., Am.J.Ophthalmol.154(2):403-412.e4(2012)). Although these diseases have different underlying disease processes, their ultimate outcome is the same: loss of RPE cells, ultimately leading to blindness.

[0096] There is an unmet medical need for therapeutic agents to reconstitute RPE and PR. Transplantation of replacement RPE cells is an attractive therapeutic paradigm because it addresses common endpoints for all these conditions. The use of patient-specific iPSC-derived RPE offers further advantages because the transplanted cells are precisely matched to the patient. In some embodiments, the method may involve harvesting patient skin cells, then reprogramming the cells to a pluripotent state using a combination of transcription factors (Yang et al., N.Engl.J.Med.359(14):1456-63(2008)), and then differentiating the iPSCs into patient-specific RPE cells for transplantation. The generation of RPE cells and their in vitro and in vivo characterization are well established. (Schwartz et al., Lancet 379(9817):713-20(2012); Abe et al., Curr. Eye Res. 20(4):268-75(2000); Carr et al., PLoS One 4(12):e8152(2009); Kamao et al., Stem Cell Reports 2(2):205-18(2014); Lu et al., Stem Cells 27(9):2126-35(2009); Lund et al., Cloning Stem Cells 8(3):189-99(2006)). RPE cells derived from hESCs and iPSCs have been shown to share similar morphological and functional characteristics. (Riera et al., Mol. Ther. Methods Clin. Dev. 3:16010(2016)). Several reports on the functional outcomes of subretinal transplantation of RPE cells in animal models of retinal degeneration (Carr et al., PLoS One 4(12):e8152(2009); Kamao et al., Stem Cell Reports 2(2):205-18(2014); Li et al., Mol.Med.18(1):1312-9(2012) and approved clinical trials for RPE cell replacement therapy have been described. However, there is still a need for effective RPE cell therapy. The methods and compositions of this disclosure can be used to address such unmet medical needs.

[0097] Method for producing cell preparations In some embodiments, the methods of the present disclosure include providing or producing induced pluripotent stem cells (iPSCs) and subsequently differentiating such iPSCs into RPE cells.

[0098] Creation of iPSCs In some embodiments, the methods of the present disclosure include obtaining and / or producing induced pluripotent stem cells (iPSCs). iPSCs are a type of pluripotent stem cell artificially induced from non-pluripotent cells such as adult cells (e.g., fibroblasts or other suitable somatic cells) by inducing the expression of specific genes. Various preferred methods for producing iPSCs are known in the art (see, for example, Takahashi et al., Cell 126:663-676 (2006); Seki et al., World J. Stem Cells 7(1):116-125 (2015); and Lakshmipathy and Vermuri, editors, Methods in Molecular Biology: Pluripotent Stem Cells, Methods and Protocols, Springer 2013). In some embodiments, iPSCs can be induced by transfection of non-pluripotent cells, such as adult fibroblasts, with specific stem cell-related genes (e.g., Oct-3 / 4 (Pouf51) and Sox-2). In some examples, transfection can be achieved by viral vectors such as retroviruses, lentiviruses, or adenoviruses. Further suitable reprogramming methods include the use of vectors that are not integrated into the host cell genome, such as episomatic vectors, or the direct delivery of reprogramming factors by RNA encoding or as proteins. For example, cells can be transfected with Oct-3 / 4, Sox-2, Klf4, and / or c-Myc using retroviral systems, or with Oct-4, Sox-2, NANOG, and / or LIN28 using lentiviral systems. In some embodiments, reprogramming can be induced by non-viral introduction of reprogramming factors, for example by introducing the protein itself, or by introducing nucleic acids encoding the reprogramming factors, for example by introducing messenger RNA that generates the reprogramming factors during translation (see, e.g., Warren et al., Cell Stem Cell, 2010 Nov. 5;7(5):618-30).Numerous suitable methods for reprogramming are known to those skilled in the art, and this disclosure is not limited thereto. Further methods and systems are commercially available (e.g., Simplicon® RNA Reprogramming Kit, EMD Millipore).

[0099] In some embodiments, iPSCs may originate from somatic cells. Somatic cells, as the term is used herein, refer to the cells that make up the body of an organism, excluding germline cells. All cell types in the mammalian body (apart from sperm and eggs, the cells from which they are produced (gamete cells) and undifferentiated stem cells) are differentiated somatic cells. For example, viscera, skin, bone, blood, and connective tissue are all composed of differentiated somatic cells. In some embodiments, the mature cells from which iPSCs are produced include somatic cells. In some embodiments, such somatic cells may be or include blood cells, such as blood mononuclear cells, e.g., whole blood mononuclear cells or peripheral blood mononuclear cells (PBMCs). In some embodiments, the mature cells from which iPSCs are produced may be or include B lymphocytes (B cells), T lymphocytes (T cells), fibroblasts, keratinocytes, etc.

[0100] In some embodiments, suitable somatic cell types for use in accordance with this disclosure include fibroblasts (e.g., primary fibroblasts), muscle cells (e.g., myocytes), cumulus cells, nerve cells, mammary gland cells, hepatocytes, and islet cells. In some embodiments, the somatic cells are primary cell lines or descendants of primary or secondary cell lines. In some embodiments, the somatic cells are obtained from human samples, such as hair follicles, blood samples, biopsies (e.g., skin biopsies or fat biopsies), swab samples (e.g., oral swab samples), and are therefore human somatic cells.

[0101] Some non-limiting examples of differentiated somatic cells include, for example, epithelial cells, endothelial cells, nerve cells, adipocytes, cardiac cells, skeletal muscle cells, skin cells, immune cells, liver cells, spleen cells, lung cells, peripheral circulating blood cells, gastrointestinal cells, kidney cells, bone marrow cells, and pancreatic cells. In some embodiments, somatic cells may be primary cells isolated from any somatic tissue, including but not limited to the brain, liver, intestines, stomach, fat, muscle, uterus, skin, spleen, endocrine organs, bone, blood, etc. In various embodiments, somatic cells may originate from mammalian species, non-limiting examples including mouse, cattle, monkey, pig, horse, sheep, or human. In some embodiments, somatic cells are human somatic cells. In preferred embodiments, somatic cells are autologous human somatic cells.

[0102] Reprogrammed somatic cells include, for example, alkaline phosphatase (AP); ABCG2; stage-specific embryonic antigen-1 (SSEA-1); SSEA-3; SSEA-4; TRA-1-60; TRA-1-81; Tra-2-49 / 6E; ERas / ECAT5, E-cadherin; β-III-tubulin; alpha-smooth muscle actin (α-SMA); fibroblast growth factor 4 (Fgf4), Cripto, Dax1; zinc finger protein 296 (Zfp296); N-acetyltransferase-1 (Nat1); (ES cell-associated transcript 1 (ECAT1)); ESG1 / DPPA5 / ECAT2; ECAT3; ECAT6; ECAT7; ECAT8; ECAT9; EC Any number of pluripotent cell markers can be expressed, including AT10; ECAT15-1; ECAT15-2; Fth117; Sal14; undifferentiated germinal cell transcription factor (Utf1); Rex1; p53; G3PDH; telomerase including TERT; silent X chromosome gene; Dnmt3a; Dnmt3b; TRIM28; F-box containing protein 15 (Fbx15); Nanog / ECAT4; Oct3 / 4; Sox2; Klf4; c-Myc; Esrrb; TDGF1; GABRB3; Zfp42; FoxD3; GDF3; CYP25A1; developmental pluripotency-associated 2 (DPPA2); T-cell lymphoma breakpoint 1 (Tcl1); DPPA3 / Stella; DPPA4, etc. Other markers may include Dnmt3L; Sox15; Stat3; Grb2; β-catenin; and Bmi1.

[0103] In some embodiments, iPSCs are derived from human fibroblasts, e.g., human skin biopsy material. Methods for producing iPSCs from human fibroblasts are known in the art (e.g., Hazim et al., Stem Cell Res. Ther. 8(1):217 (2017); Hazim et al., Stem Cell Res. Ther. 10(1):52 (2019)). In some embodiments, human fibroblasts are isolated from human skin biopsies, expanded in cell culture, and characterized for the expression of one or more fibroblast markers (e.g., fibroblast-specific protein-1 (FSP-1) and / or vimentin (VIM)) by immunostaining, for example. In some embodiments, cultured fibroblasts are selected for one or more further processing steps (e.g., cryopreservation and / or reprogramming into iPSCs) if they are double-positive for FSP-1 and VIM. In some embodiments, fibroblasts are selected for cryopreservation if they are double-positive for FSP-1 and VIM. In some embodiments, cryopreserved fibroblasts are thawed using, for example, the method described herein, before reprogramming into iPSCs. In some embodiments, iPSCs are characterized for the expression of one or more pluripotency markers, for example, one or more pluripotency markers described herein, for example, SSEA-4 and / or Oct-4. In some embodiments, iPSCs are selected for one or more further processing steps (e.g., cryopreservation and / or differentiation into RPE cells as described herein) if they express one or more pluripotency markers, for example, one or more pluripotency markers described herein, for example, if they are double-positive for SSEA-4 and Oct-4.

[0104] Preparation of RPE cell preparations In some embodiments, the methods of the present disclosure include generating RPE cells, for example, differentiating iPSCs into RPE cells. Specific methods for differentiating iPSCs into RPE cells are known in the art (e.g., Hazim et al., Stem Cell Res. Ther. 8(1):217 (2017); Hazim et al., Stem Cell Res. Ther. 10(1):52 (2019)). In some embodiments, differentiation into RPE cells is initiated by replacing the pluripotency-supporting culture medium with a differentiation medium containing one or more differentiation-inducing factors, such as activin, a nodal signaling inhibitor, a Wnt signaling inhibitor, and / or a sonic hedgehog signaling inhibitor.

[0105] In some embodiments, the differentiation medium comprises Dulbecco's modified Eagle medium:nutrient mixture F-12 (DMEM / F12) supplemented with xeno-free knockout serum (Invitrogen), NEAA, glutamine, and / or nicotinamide. In some embodiments, the differentiation medium may include one or more additional differentiation agents, such as members of the transforming factor-β (TGFβ) superfamily (e.g., activin A, activin B, and activin AB), Nodal, anti-Müllerian hormone (AMH), bone morphogenetic proteins (BMPs) (e.g., BMP2, BMP3, BMP4, BMP5, BMP6, and BMP7, growth and differentiation factors (GDF)), WNT pathway inhibitors (e.g., CKI-7, DKK1), TGF pathway inhibitors (e.g., LDN193189, Noggin), BMP pathway inhibitors (e.g., SB431542), sonic hedgehog signaling inhibitors, bFGF inhibitors, and / or MEK inhibitors (e.g., PD0325901).

[0106] In some embodiments, iPSCs differentiate into RPE cell lines in a first differentiation medium containing a first differentiation agent, and then further differentiate into RPE cells in a second differentiation medium containing a second differentiation agent. In some embodiments, the first differentiation medium contains nicotinamide (e.g., about 5 mM nicotinamide to about 20 mM nicotinamide, e.g., about 10 mM nicotinamide), and the second differentiation medium contains activin (e.g., activin A) (e.g., about 100 ng / ml of activin A to about 300 ng / ml of activin A, e.g., about 140 ng / ml of activin A). In some embodiments, the first differentiation medium comprises DMEM / F12 supplemented with approximately 10% to approximately 20% (e.g., approximately 14%) xeno-free knockout serum, approximately 0.05 mM to approximately 5 mM (e.g., approximately 0.1 mM) of NEAA, approximately 0.5 mM to approximately 5 mM (e.g., approximately 2 mM) of L-glutamine, and approximately 5 mM to approximately 20 mM (e.g., approximately 10 mM) of nicotinamide. The second differentiation medium comprises DMEM / F12 supplemented with activin A (e.g., approximately 100 ng / ml to approximately 300 ng / ml of activin A, e.g., approximately 140 ng / ml of activin A) and approximately 5 ng / ml to approximately 50 ng / ml (e.g., approximately 20 ng / ml) of basic fibroblast growth factor (e.g., FGF2). In some embodiments, the differentiation medium is changed daily or every 2-3 days during differentiation (e.g., in the first differentiation medium and / or the second differentiation medium). In some embodiments, cells are cultured in the first differentiation medium and / or the second differentiation medium for about 3-12 weeks, e.g., 6-10 weeks, 2-8 weeks, or 3-6 weeks. In some embodiments, cells are cultured in the first differentiation medium for about 2 weeks and then in the second differentiation medium for about 2 weeks.

[0107] In some embodiments, after cells have been cultured in a first and / or second differentiation medium, the cells are then cultured in DMEM / F12 basal medium until, for example, pigmentation is detected (for example, until a large percentage of the cells, e.g., about 50%, 60%, 70%, 80%, 85%, 90%, 95%, 97%, or more, are determined to be pigmented using visual analysis, such as using a phase-contrast microscope). In some embodiments, pigment cells are differentially passaged using RPE medium containing DMEM / F12 supplemented with, for example, a dissociation reagent (e.g., trypsin, e.g., TrypLE), as well as fetal bovine serum (FBS) (e.g., about 2% to about 8%, e.g., about 5% FBS), human AB serum (e.g., about 2% to about 6%, e.g., about 4% human AB serum), taurine (e.g., about 0.05 to about 0.5 mg / ml, e.g., about 0.25 mg / ml taurine), nicotinamide (e.g., about 5 mM to about 20 mM, e.g., 10 mM nicotinamide), NEAA, N2, B27, beta-mercaptoethanol (e.g., about 0.05 mM to about 0.5 mM, e.g., about 0.1 mM beta-mercaptoethanol), and 1×Glutamax®. In some embodiments, the pigment cells are mechanically passaged by harvesting the cells in culture medium after a TrypLE treatment of approximately 5, 8, 10, 12, or 15 minutes. In some embodiments, the cells are differentially passaged at least once, at least twice, at least three times, at least four times, at least five times, at least six times, or at least eight times.

[0108] In some embodiments, the presence of one or more morphological quality control criteria, e.g., one or more morphological parameters from the following (confluent hexagonal cells, pigmentation, cobblestone pattern, and / or well-defined membrane boundaries (see, e.g., Figure 2)) is evaluated, and the timing of the passage step (e.g., duration of treatment with a dissociation reagent, e.g., trypsin treatment) and / or the number of passage steps is determined if one or more of the confluent hexagonal cells, pigmentation, cobblestone pattern, and / or well-defined membrane boundaries are met. In some embodiments, after one passage step, the cells are evaluated for one or more morphological parameters. In some embodiments, if, after one passage step, the cells do not meet the morphological quality control criteria (e.g., at least 80%, 85%, 90%, or 95% of the cells do not exhibit one or more morphological parameters), the cells are subjected to at least one additional passage step. In some embodiments, if, after one passaging step, the cells do not meet the morphological quality control criteria (e.g., at least 80%, 85%, 90%, or 95% of the cells do not exhibit all morphological parameters), the cells are subjected to at least one additional passaging step. In some embodiments, the passaging step is repeated until the morphological quality control criteria are met (e.g., at least 80%, 85%, 90%, or 95% of the cells exhibit one or more morphological parameters). In some embodiments, the passaging step is repeated until the morphological quality control criteria are met (e.g., at least 80%, 85%, 90%, or 95% of the cells exhibit all morphological parameters).

[0109] In some embodiments, cells are passaged as described herein and that meet such morphological quality control criteria also meet one or more of the following functional quality control criteria: (i) RPE cells have at least about 50 ohms / cm² after, for example, 1–8 weeks of culture. 2 , at least about 75 ohms / cm 2 at least approximately 100 ohms / cm² 2, at least about 125 ohms / cm 2 , at least about 150 ohms / cm 2 , at least about 175 ohms / cm 2 , or at least about 200 ohms / cm² 2 (ii) cells exhibit differential secretion of VEGF at a ratio of basal secretion to apical secretion of at least approximately 0.25, at least approximately 0.5, at least approximately 0.75, at least approximately 1, at least approximately 1.25, or at least approximately 1.5; (iii) cells exhibit differential secretion of PEDF at a ratio of apical secretion to basal secretion of at least approximately 0.25, at least approximately 0.5, at least approximately 0.75, at least approximately 1, at least approximately 1.25, or at least approximately 1.5; and / or (iv) at least approximately 50%, approximately 60%, approximately 70%, approximately 80%, approximately 90%, or approximately 95% of RPE cells exhibit the ability to phagocytose POR (e.g., as assessed using flow cytometry).

[0110] In some embodiments, RPE cells that meet one or more such quality control criteria (e.g., one or more morphological quality control criteria described herein and / or one or more functional quality control criteria described herein) are selected for one or more further processing steps (e.g., expansion, preparation into RPE cell preparations, e.g., RPE cell drug substance, and / or cryopreservation). In some embodiments, RPE cells that meet all of the morphological quality control criteria described herein and all of the functional quality control criteria described herein are selected for one or more further processing steps (e.g., expansion to achieve a predetermined number of cells, preparation into RPE cell preparations, e.g., preparation as RPE cell drug substance, and / or cryopreservation). In some embodiments, RPE cells that meet all of the morphological quality control criteria described herein and all of the functional quality control criteria described herein are expanded to achieve a predetermined number of cells, prepared into RPE cell preparations (e.g., RPE cell drug substance), and cryopreserved.

[0111] Production of RPE cell products In some embodiments, the methods of the present disclosure include preparing an RPE cell product (e.g., an RPE cell drug) from an RPE cell preparation (e.g., an RPE cell drug) prepared, for example, as described herein. In some embodiments, the RPE cell preparation (e.g., an RPE cell drug) has undergone at least one freeze / thaw cycle. Certain aspects of the present disclosure are based on the discovery that it is beneficial to culture such a previously cryopreserved and frozen RPE cell preparation, for example, before administration to a therapeutic formulation and / or subject. For example, culturing such a previously cryopreserved and frozen RPE cell preparation can improve the viability and / or potency of such cells. In some embodiments, the frozen RPE cell preparation (e.g., a frozen RPE cell drug) is thawed and the cells are cultured in a suitable cell culture medium for about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, or about 6 weeks. In some embodiments, after such a culture step, the RPE cells have viability of at least about 80%, 85%, 90%, or 95% compared to a reference preparation of RPE cells that have not undergone at least one freeze / thaw cycle, e.g., an RPE cell pharmaceutical described herein. In some embodiments, after such a culture step, at least 80%, 85%, 90%, 95%, or higher percentages of the RPE cells are viable. In some embodiments, such cultured RPE cells are formulated as a therapeutic composition, e.g., an RPE cell pharmaceutical.

[0112] In some embodiments, a method for producing an RPE cell drug may comprise four steps, as schematically shown in Figure 1, for example. As shown in Figure 1, step 1 may comprise isolating fibroblasts from a skin biopsy of interest, expanding the isolated fibroblasts, preserving the fibroblasts, and testing the preserved fibroblasts as may be described herein. As shown in Figure 1, step 2 may comprise thawing the preserved fibroblasts, reprogramming the fibroblasts into iPSCs, expanding the iPSCs, preserving the iPSCs, and testing the preserved iPSCs using one or more criteria as may be described herein. As shown in Figure 1, step 3 may comprise thawing the preserved iPSCs, differentiating the iPSCs into RPE cells, expanding the RPE cells, preserving the RPE cells (e.g., as an RPE cell drug substance (DS)), and testing the preserved RPE cells using one or more criteria as may be described herein. As shown in Figure 1, step 4 may include thawing the stored RPE cells (DS), culturing the thawed RPE cells for about 4 weeks, testing the RPE cells using one or more criteria described herein, preparing doses of the RPE cell preparation, performing dose-release testing using one or more criteria described herein, and producing the RPE cell drug (DP). The durations of the various steps shown in Figure 1 are illustrative, and in some embodiments, the duration of one or more steps shown in Figure 1 may be shorter or longer than the times shown in Figure 1. For example, while the exemplary method in Figure 1 takes place over a period of about 1 month, in some embodiments, step 1 may be shorter (e.g., about 1 week, 2 weeks, 3 weeks, or 3.5 weeks) or longer (e.g., about 4.5 weeks, 5 weeks, 6 weeks, 7 weeks, or 8 weeks).Additionally or alternatively, while the exemplary method in Figure 1 shows that the duration of Stage 2 is approximately 4 months, in some embodiments, Stage 2 may be shorter (e.g., about 1 month, 2 months, 3 months, or 3.5 months) or longer (e.g., about 4.5 months, 5 months, 6 months, 7 months, or 8 months). Additionally or alternatively, while the exemplary method in Figure 1 shows that the duration of Stage 3 is approximately 5 months, in some embodiments, Stage 3 may be shorter (e.g., about 1 month, 2 months, 3 months, 4 months, or 4.5 months) or longer (e.g., about 5.5 months, 6 months, 7 months, or 8 months). Additionally or alternatively, while the exemplary method in Figure 1 shows that the duration of Stage 4 is approximately 1 month, in some embodiments, Stage 4 may be shorter (e.g., about 1 week, 2 weeks, 3 weeks, or 3.5 weeks) or longer (e.g., about 4.5 weeks, 5 weeks, 6 weeks, 7 weeks, or 8 weeks). In some embodiments, the duration of one or more stages shown in Figure 1 is determined using the quality control criteria described herein (e.g., morphological and / or functional parameters).

[0113] Methods for evaluating cells In some embodiments, the methods of the present disclosure include evaluating cells, for example, iPSCs and / or RPE cells as described herein. In some embodiments, the cells are, for example, alkaline phosphatase (AP); ABCG2; stage-specific embryonic antigen-1 (SSEA-1); SSEA-3; SSEA-4; TRA-1-60; TRA-1-81; Tra-2-49 / 6E; ERas / ECAT5, E-cadherin; β-III-tubulin; alpha-smooth muscle actin (α-SMA); fibroblast growth factor 4 (Fgf4), Cripto, Dax1; zinc finger protein 296 (Zfp296); N-acetyltransferase-1 (Nat1); (ES cell-associated transcript 1 (ECAT1)); ESG1 / DPPA5 / ECAT2; ECAT3; ECAT6; ECAT7; ECAT8; ECAT9; ECAT10; ECAT15- The expression of one or more pluripotent cell markers, including 1;ECAT15-2;Fth117;Sal14;Undifferentiated germinal cell transcription factor (Utf1);Rex1;p53;G3PDH;Telomerase including TERT;Silent X chromosome gene;Dnmt3a;Dnmt3b;TRIM28;F-box containing protein 15 (Fbx15);Nanog / ECAT4;Oct3 / 4;Sox2;Klf4;Lin28,c-Myc;Esrrb;TDGF1;GABRB3;Zfp42,FoxD3;GDF3;CYP25A1;Developmental pluripotency-related 2 (DPPA2);T-cell lymphoma breakpoint 1 (Tcl1);DPPA3 / Stella; and / or DPPA4, can be evaluated. Other markers may include Dnmt3L;Sox15;Stat3;Grb2;β-catenin and / or Bmi1. In some embodiments, cells can be evaluated for the expression of one or more RPE cell markers such as RPE65, CRALBP, PEDF, bethrophilin-1 (BEST1), RLBP1, merTK, MITF, OTX2, PAX2, PAX6, premelanosome proteins (gp-100 or PMEL, e.g., PMEL-17), CD140b, tyrosinase and / or ZO1. In some embodiments, cells can be evaluated for the expression of non-RPE lineage markers such as GD2 and / or CD184.Methods for evaluating the expression of such markers are well known in the art, and include, for example, flow cytometry, immunohistochemistry, PCR, ddPCR, and next-generation sequencing.

[0114] In some embodiments, cells are evaluated for the presence of one or more morphological features (e.g., morphological parameters described herein). Methods for evaluating such morphological features are known in the art, such as visual evaluation using microscopy. In some embodiments, cells are evaluated for viability using methods known in the art, for example, trypan blue staining.

[0115] In some embodiments, cells are evaluated for one or more functional parameters, such as those described herein. For example, transepithelial electrical resistance (TEER) can be measured, for example, to evaluate barrier function, using methods known in the art (see, for example, Markert et al., Front.Cell Dev.Biol.10:910040(2022); Hazim et al., Stem Cell Res.Ther.8(1):217(2017)). Devices for measuring TEER are known in the art, for example, EVOM 2 An epithelial volt-ohmmeter (World Precision Instruments) is one example. In some embodiments, the efficacy of cells is evaluated by analyzing polarized PEDF and VEGF secretion using methods known in the art, for example. For example, RPE cells can be cultured on a Matrigel-coated Transwell membrane, apical and basal media can be collected, and PEDF and VEGF can be measured by ELISA (e.g., Zhang et al., Stem Cell Res. Ther. 13(1):454 (2022)). The ability of RPE cells to phagocytose photoreceptor outer segments can be measured using methods known in the art (e.g., Hazim et al., Stem Cell Res. Ther. 8(1):217 (2017)).

[0116] RPE cell preparation In some embodiments, the present disclosure provides RPE cell preparations, such as RPE cell drug substances and / or RPE cell pharmaceuticals, that meet certain criteria and / or are defined by certain characteristics. In some embodiments, RPE cell preparations, e.g., RPE cell drug substances and / or RPE cell pharmaceuticals, satisfy one or more of the following criteria: (1) at least 80%, 85%, 90%, 95%, or 98% of the RPE cells exhibit confluent hexagonal cells, pigmentation, cobblestone morphology, and well-defined membrane boundaries; (2) gene expression of RPE65 ≥ 0.5 as assessed by ddPCR; (3) gene expression of BEST1 ≥ 6 as assessed by ddPCR; (4) gene expression of RLBP ≥ 1 as assessed by ddPCR; (5) gene expression of MerTK ≥ 0.15 as assessed by ddPCR; (6) expression of MiTF, BEST1, and / or ZO-1 at the same level as in primary RPE cell lines, or in primary RPE cell lines (7) The expression of MiTF, BEST1 and / or ZO-1 differs by only about 1%, 5%, or 10% or less; (8) At least about 70%, 80%, 85%, or 90% of the cells are viable for up to about 6 hours after passage via, for example, an implantation device (e.g., a 41g device); (9) At least about 70%, 80%, 85%, or 90% of the cells express PMEL17; (10) Approximately 0% when evaluated by, for example, ddPCR. (10) Less than 0.005%, less than 0.004%, less than 0.003%, or less than 0.002% of cells are positive for Lin28, Oct-4, and / or Klf4 expression; (11) At least about 80%, 85%, or 90% of cells express PMEL17 and CD140b, but not CD184 or GD2 (PMEL17+CD140b+CD184-GD2-); (12) RPE cells have at least about 50 ohms / cm² 2 , at least about 75 ohms / cm 2 at least approximately 100 ohms / cm² 2 , at least about 125 ohms / cm 2 , at least about 150 ohms / cm2 , at least about 175 ohms / cm 2 , or at least about 200 ohms / cm² 2 The TEER is, for example, shown between 1 and 8 weeks in culture, (12) the cells show differential secretion of VEGF at a ratio of basal secretion to apical secretion of at least about 0.25, at least about 0.5, at least about 0.75, at least about 1, at least about 1.25, or at least about 1.5, (13) the cells show differential secretion of PEDF at a ratio of apical secretion to basal secretion of at least about 0.25, at least about 0.5, at least about 0.75, at least about 1, at least about 1.25, or at least about 1.5, and / or (14) at least about 50%, about 60%, about 70%, about 80%, about 90%, or about 95% of the RPE cells show the ability to phagocytose POR (e.g., as assessed using flow cytometry). In some embodiments, RPE cell preparations, e.g., RPE cell active pharmaceutical ingredients and / or RPE cell pharmaceuticals, satisfy at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, or 14 of these criteria. In some embodiments, RPE cell preparations, e.g., RPE cell active pharmaceutical ingredients and / or RPE cell pharmaceuticals, have undergone at least one freeze / thaw cycle and satisfy at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, or 14 of these criteria. In some embodiments, RPE cell preparations, e.g., RPE cell drug substance and / or RPE cell pharmaceuticals, undergo at least one freeze / thaw cycle and are then cultured for about 1, 2, 3, or 4 weeks or longer, satisfying at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, or 14 of these criteria.

[0117] In some embodiments, the methods of this disclosure have various applications, including, for example, quality control at different stages of manufacturing, analysis of RPE cell preparations before or after completion of manufacturing (e.g., before or after distribution to a filling / finishing environment or facility), and before or after commercialization (e.g., before distribution to a pharmacy, caregiver, patient, or other end-user). Thus, the preparation may be any preparation potentially containing an RPE cell active pharmaceutical ingredient or an RPE cell medicinal product. In some embodiments, the preparation is from a production or use stage before release to a caregiver or other end-user, before packaging into individual dosage forms such as syringes, pens, vials, or multi-dose vials, before it is determined that the batch can be commercially released, and before a test certificate, material safety data sheet (MSDS), or certificate of analysis (CofA) for the preparation is prepared. In some embodiments, the RPE cell preparation is from, for example, during the differentiation stage from iPSCs, during the RPE cell expansion stage, or during the RPE cell medicinal product production stage. In some embodiments, the RPE cell preparation is from stage 3 or stage 4, as shown in Figure 1.

[0118] Evaluations from the methods of this disclosure are useful, for example, to induce, control, or carry out certain activities or steps in the processes of preparation, distribution, monitoring, and delivery for the safe and effective use of RPE cell drug substances and / or RPE cell pharmaceuticals. Thus, in some embodiments, for example, decisions or steps are made in response to the evaluation, for example, depending on whether criteria are met. The methods may further include either or both decisions to take a step and / or decisions to carry out the step itself. For example, the steps may include: classifying the preparation (or another preparation in which the preparation is representative), selecting it, accepting or discarding it, releasing it into a pharmaceutical product or processing it, making it unsuitable for commercial release (e.g., labeling, isolating, or destroying it), passing it on to subsequent steps in manufacturing, reprocessing it (e.g., the preparation undergoes a repeat of a previous processing step or is subjected to a modification process), formulating it into an active pharmaceutical ingredient or pharmaceutical product (e.g., combining it with another component (e.g., an excipient, buffer, or diluent)), placing it in a container, dividing it into smaller aliquots (e.g., unit dose or multi-dose containers), packaging it, shipping it, moving it to another location, combining it with another element to form a kit, combining it (e.g., packaging it with a delivery device, diluent, or instruction manual), commercial release, selling it or offering it for sale, delivering it to a caregiver or other end-user, or administering it to a target. For example, based on the result of a decision or whether one or more criteria are met, or in comparison to a reference standard, batches from which the preparation is drawn can be processed, for example, as described above.

[0119] Methods described herein may include making a determination of (a) whether the preparation can be formulated into an active pharmaceutical ingredient or a medicinal product, (b) whether the preparation can be reprocessed (for example, the preparation may undergo a repetition of the previous process step), or (c) whether the preparation is unsuitable for formulation into an active pharmaceutical ingredient or a medicinal product. In some cases, the method includes making the preparation unsuitable for commercial release by formulating as referred to in step (a), reprocessing as referred to in step (b), or by labeling or destroying the preparation, for example, as referred to in step (c).

[0120] Disability / Illness The RPE cell preparations described herein can be used in cell-based treatments where RPE cells are required or to improve treatment. In some embodiments, the provided preparations may be used to treat ocular pathologies. In some embodiments, the methods of using RPE cells described herein can be used to treat various conditions, for example, as described in U.S. Patent No. 10,077,424.

[0121] Specific treatment regimens, routes of administration, and any adjunctive therapies can be adjusted according to appropriate medical practice, for example, based on the specific condition, severity, and overall health status of the subject. In some embodiments, RPE cell administration may be effective in completely restoring vision loss or other symptoms. In some embodiments, RPE cell administration may be effective in reducing the severity of symptoms and / or preventing further degeneration of the subject's condition. Alternatively or additionally, in some embodiments, RPE cell administration can be used to help treat symptoms of any damage to the endogenous RPE layer or any other ophthalmic layer. In some embodiments, the RPE cell preparations described herein can be used to treat age-related macular degeneration, myopic degeneration, Stargardt disease, choroidal neovascularization, any condition, or diseases resulting in scarring, atrophy, or dysfunction of the RPE layer. In some embodiments, the preparations provided are used to treat conditions or diseases that cause retinal atrophy or edema or degeneration or detachment. In some embodiments, the preparations provided are used to treat conditions or diseases that result in photoreceptor loss or dysfunction. In some embodiments, the provided preparations are used to treat conditions or diseases affecting the corneal endothelium or corneal epithelium. In some embodiments, the provided preparations are used to treat ocular pathologies.

[0122] In some embodiments, this disclosure provides a method for treating a disease or disorder of the retina by administering the RPE preparation described herein to the eye of interest. In some embodiments, the diseases or disorders of the retina include, for example, retinal degeneration, such as age-related macular degeneration (dry or wet), retinal detachment, retinitis pigmentosa, Stargardt disease, myopic degeneration, Best's disease, and macular telangiectasia.

[0123] Age-related macular degeneration (AMD) is a progressive degenerative disease that is the leading cause of vision loss in the elderly population. Degeneration / dysregulation of the retinal pigment epithelium (RPE), the supporting monolayer of cells beneath the photoreceptors, is commonly seen in AMD patients. While treatments exist for the neovascular / exudative form of AMD, there are currently no treatments for the non-exudative / dry form of AMD (Somasundaran S, Constable IJ, Mellow CB, Carvalho LS. Retinal pigment epithelium and age-related macular degeneration: A review of major disease mechanisms. Clin Exp Ophthalmol. 2020 Nov;48(8):1043-1056. doi:10.1111 / ceo.13834. Epub 2020 Aug 17. PMID:32710488;PMCID:PMC7754492).

[0124] In some embodiments, the Disclosure provides a method for treating disorders associated with retinal degeneration, including macular degeneration, by administering the RPE cell preparation described herein to a target eye. In some embodiments, the Disclosure provides a method for treating eye diseases, including hereditary and acquired eye diseases, by administering the RPE cell preparation described herein to a target eye. Examples of acquired or hereditary eye diseases include age-related macular degeneration, myopic degeneration, Stargardt disease, choroidal neovascularization, any condition or disease resulting in scarring or atrophy or dysfunction of the RPE layer, any condition or disease resulting in retinal atrophy or edema or degeneration or detachment, any condition or disease resulting in loss or dysfunction of photoreceptors, any condition or disease affecting the corneal endothelium or epithelium, or any other eye condition.

[0125] Pathological myopia represents a subgroup of myopia affecting up to 3% of the world's population. The visual loss associated with pathological myopia can be progressive and / or irreversible, making it clinically significant as it impacts individuals during their most productive years. Progressive thinning and attenuation of the retinal pigment epithelium (RPE) occurs at various clinical stages throughout the fundus. RPE atrophy and the distribution of variable light reflection can also be observed in younger patients with high myopia. Patients with pathological myopia present with myopic maculopathy of comparable or greater severity than diffuse chorioretinal atrophy. Myopic maculopathy includes diffuse chorioretinal atrophy, maculine chorioretinal atrophy, lacquer cracks, myopic choroidal neovascularization (myopic CNV), and CNV-associated macular atrophy. Currently, there are no known topical, local, or systemic drug therapies or surgeries that effectively modify retinochoroidal and RPE atrophy. Patients developing myopic CNV can be treated with anti-VEGF agents. (Ryan et al., Retina. 2013, Ohno-Matsui K. Pathologic myopia. Asia Pac J Ophthalmol 2016;5:415-423).

[0126] Stargardt disease is the most common form of juvenile macular degeneration, characterized by progressive loss of central vision, with symptom onset typically occurring during adolescence. Affected individuals have mutations in the ABCA4 gene, which encodes the adenosine triphosphate (ATP)-binding cassette transporter for photoreceptors. When functional, ABCA4 helps remove all-trans retinaldehyde, thus reducing the excessive accumulation of toxic bisretinoid fluorophores that make up lipofuscin in the retina. Reduced or absent transporter activity, such as STGD1, leads to a more rapid accumulation of lipofuscin in retinal pigment epithelial (RPE) cells, the ultimate consequence of which is RPE and photoreceptor cell degeneration. Currently, there are no available treatments for Stargardt disease.

[0127] Formulation and administration In some embodiments, the RPE cells described herein are delivered or administered to the eye of a subject in a pharmaceutically acceptable ophthalmic formulation, for example, by intraocular injection or application to the ocular surface. In some embodiments, the cell preparation provided is administered subretinally. The concentration for injection may be any amount that is effective and non-toxic. In some embodiments, about 10,000 to about 1,000,000 (e.g., about 10,000 to about 5,000) RPE cells are administered to the subject. In some embodiments, about 10,000, 30,000, 50,000, 100,000, 150,000, 200,000, 250,000, 300,000, 350,000, 400,000, 450,000, or 500,000 to 1,000,000 or more RPE cells are administered to the subject. In some embodiments, the cells are administered as a single dose. In some embodiments, the cells are administered in multiple doses.

[0128] In some embodiments, RPE cells are formulated for delivery in a pharmaceutically acceptable ophthalmic vehicle. In some embodiments, such a vehicle maintains a preparation of RPE cells in contact with the ocular surface for a period of time sufficient for the cells to penetrate the affected area of ​​the eye, e.g., the anterior chamber, posterior chamber, vitreous body, aqueous humor, vitreous fluid, cornea, iris / ciliary body, lens, choroid, retina, sclera, epichoroidal space, conjunctiva, subconjunctival space, episcleral space, intracorneal space, epicorneal space, subcorneal space / corneal endothelial layer, ciliary body squamata, surgically induced avascular area, macula, and any retinal area. The Disclosure further includes products and systems, such as delivery vehicles containing the RPE cells described herein, as well as kits containing such delivery vehicles and / or systems.

[0129] In some embodiments, the RPE cell preparations described herein are administered to the eye of interest using an implant or device. In certain embodiments, the device is a biodegradable implant for treating an eye condition, comprising, for example, RPE cells dispersed in a biodegradable polymer matrix. In some embodiments, the device is or comprises particles (e.g., a polymer matrix, e.g., a biodegradable polymer matrix, or particles comprising such a matrix, wherein the RPE cells described herein are associated, e.g., dispersed therein). In some such embodiments, at least about 75% of the particles have a diameter of less than about 10 μm.

[0130] In some embodiments, implants, such as biodegradable implants, can be sized for implantation into ocular regions such as the anterior chamber, posterior chamber, vitreous cavity, choroid, suprachoroidal space, conjunctiva, subconjunctival space, episcleral space, corneal space, supracorneal space, subcorneal space / corneal endothelium, sclera, ciliary body squamata, surgically induced avascular areas, macula, and retina / subretinal space.

[0131] In some embodiments, the biodegradable polymer may be, for example, a poly(lactic acid-co-glycol) acid (PLGA) copolymer, or may include one. In certain embodiments, the ratio of lactic acid monomer to glycolic acid monomer in the polymer is about 25 / 75, about 40 / 60, about 50 / 50, about 60 / 40, about 75 / 25 weight percent, more preferably about 50 / 50. Furthermore, the PLGA copolymer may be about 20, about 30, about 40, about 50, about 60, about 70, or about 80-90% by weight of the biodegradable implant. In certain embodiments, the PLGA copolymer may be about 30-50% by weight, preferably about 40% by weight, of the biodegradable implant.

[0132] In some embodiments, the RPE cell preparations described herein are administered to the target eye on a scaffold, matrix, or bioink.

[0133] The volume of the RPE cell preparation administered according to the method described herein may depend on factors such as the mode of administration, the number of RPE cells, the age of the subject, and the type and severity of the disease being treated. In some embodiments, the RPE cell preparation is administered by infusion in liquid volumes of, for example, about 5.0 microliters to about 50 microliters, about 50 microliters to about 250 microliters, and about 250 microliters to about 1 milliliter. In some embodiments, the RPE cells are administered by infusion in a liquid volume of about 150 microliters.

[0134] In some embodiments, the RPE cell preparation is administered to the target eye by intraocular injection. The administration regimen can be adjusted to provide the optimal desired response (e.g., therapeutic response).

[0135] In some embodiments, the RPE cell preparation is administered to the subject's eye once or more periodically throughout the subject's lifetime, for example, once a year, once every 6-12 months, once every 3-6 months, once every 1-3 months, or once every 1-4 weeks. Alternatively, more frequent administration may be desirable for certain conditions or disorders.

[0136] In some embodiments, subjects treated with RPE cell preparations are also administered immunosuppressive therapy before, concurrently with, or after RPE cell administration. In some embodiments, for example, specifically, in embodiments where allogeneic cells (rather than autologous cells) are utilized, immunosuppressive therapy may be required over the lifetime of the subject or for a shorter period of time. Examples of immunosuppressive therapies include, but are not limited to, one or more of the following: anti-lymphocyte globulin (ALG) polyclonal antibodies, anti-thymocyte globulin (ATG) polyclonal antibodies, azathioprine, BASILIXIMAB® (anti-IL-2Ra receptor antibody), cyclosporine (cyclosporine A), DACLIZUMAB® (anti-IL-2Ra receptor antibody), everolimus, mycophenolate, RITUX1MAB® (anti-CD20 antibody), sirolimus, tacrolimus (Prograf®), and mycophenolate mofetil (MMF).

[0137] This disclosure includes compositions, e.g., pharmaceutical compositions, that contain the RPE cell preparations described herein, formulated with a pharmaceutically acceptable carrier. Methods well known in the art for preparing formulations can be found, for example, in "Remington: The Science and Practice of Pharmacy" (20th ed., ed. ARGennaro A R., 2000, Lippincott Williams & Wilkins, Philadelphia, Pa.). In some embodiments, the RPE cells described herein are formulated with a pharmaceutically acceptable carrier. In some embodiments, a pharmaceutical composition suitable for parenteral administration may include RPE cells combined with one or more pharmaceutically acceptable sterile isotonic aqueous or non-aqueous solutions, dispersions, suspensions or emulsions, or sterile powders that can be reconstituted into a sterile injectable solution or dispersion immediately before use, which may contain antioxidants, buffers, bacteriostatic agents, solutes that are isotonic with the blood of the recipient to whom the formulation is intended, or suspending or thickening agents. Suitable aqueous and non-aqueous carriers that can be used in the pharmaceutical compositions of the present invention include water, ethanol, polyols (e.g., glycerol, propylene glycol, polyethylene glycol, etc.), and suitable mixtures thereof. Appropriate fluidity can be maintained, for example, by the use of coatings such as lecithin, maintaining the required particle size in the case of dispersions, and by the use of surfactants.

[0138] The prevention of the presence of microorganisms can be ensured by including one or more antimicrobial and antifungal agents, such as parabens, chlorobutanol, and phenolsorbic acid.

[0139] The actual dose levels of RPE cells in the pharmaceutical compositions provided by this disclosure may be varied to obtain an amount of RPE cells, composition, and mode of administration effective in achieving a desired therapeutic response for a particular patient without being toxic to the patient. In some embodiments, the selected dose level depends on one or more of a variety of pharmacokinetic factors, including the activity of the particular composition of RPE cells, the route of administration, the time of administration, the duration of treatment, other drugs, compounds and / or materials used in combination with the particular composition used, the age, sex, weight, condition, overall health and prior medical history of the patient being treated, and similar factors well known in the medical field.

[0140] In some embodiments, the therapeutic composition can be administered with medical devices known in the art. For example, in some embodiments, RPE cells are delivered as a cell suspension using a delivery device (e.g., a needle or injection cannula) having, for example, an outer diameter of 90–100 μm. In some embodiments, RPE cells are delivered as a cell suspension using a delivery device (e.g., a needle or injection cannula) having, for example, an inner aperture diameter of 65–75 μl. In some embodiments, RPE cells are loaded into a delivery device (e.g., a 1 mL syringe) using an 18 G needle. In some embodiments, the 18 G needle can be replaced with an extension tube (e.g., 5–10 cm) through which air is removed. An injection cannula having a tip with, for example, an outer diameter of 90–100 μm (e.g., 41 G) can then be attached to the end of the extension tube. In some embodiments, the inner diameter of the tip opening is about 65–75 μm (e.g., about 70 μm). In some embodiments, the cannula includes a 41G tip (e.g., made of Peregrine). In some embodiments, the cannula is a 25G cannula. In some embodiments, the disclosure provides a product comprising an 18G needle and a 25G / 41G cannula. Such devices may be used for the uptake of RPE cells and subsequent intraocular administration of RPE cells. In some embodiments, the device may further include an extension tube (e.g., about 5 cm to 15 cm) and a syringe (e.g., a 1 to 2 ml syringe). Many other such implants, delivery systems, and modules are known to those skilled in the art. In some embodiments, RPE cells are delivered on / into a scaffold by a designated carrier.

[0141] All publications, patent applications, patents, and other references referenced herein are incorporated by reference in their entirety. Furthermore, the materials, methods, and examples are illustrative and not intended to limit the scope. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art to which the invention pertains. Similar or equivalent methods and materials to those described herein may be used in the practice or testing of this disclosure, but suitable methods and materials are described below.

[0142] This disclosure is further illustrated by the following embodiments. The embodiments are provided for illustrative purposes only and should not be construed as limiting the scope or content of this disclosure. [Examples]

[0143] Examples Example 1 - AiPSC-RPE Manufacturing Process Patient-specific autologous iPSC-derived RPE ("AiPSC-RPE") products were manufactured from patient-derived skin biopsies, with one batch produced per patient. The manufacturing process for the AiPSC-RPE active pharmaceutical ingredient (DS) and drug product (DP) involved four steps, as shown in Figure 1, and is discussed below.

[0144] Stage 1 (from skin biopsy to fibroblast bank) Fibroblasts were prepared and stored as described in Hazim et al., Stem Cell Res.Ther. 10(1):52 (2019). Briefly, punch biopsies were obtained from subjects and immediately immersed in Dulbecco's Modified Eagle Medium (DMEM) / F12. A pellet containing dissociated cells and tissue aggregates was collected. The medium was changed every 72 hours until the cell monolayer reached 70% confluence, and then the cells were passaged using TrypLE. Once the cells were confluent, they were passaged and characterized by immunostaining for fibroblast markers (fibroblast-specific protein-1 (FSP-1) and vimentin) to confirm their identity. Subsequently, single-cell suspensions with viability exceeding 70% were cryopreserved in ProFreeze's chemically defined freezing medium (CDM) according to the manufacturer's protocol. A summary of the results of the characterization of the fibroblast cell lines is shown in Table 1. [Table 1]

[0145] Stage 2 (From thawing fibroblasts in the fibroblast bank to the iPSC bank) iPSCs were prepared and stored as described in Hazim et al., Stem Cell Res. Ther. 10(1):52 (2019). Briefly, patient-specific fibroblasts were thawed and then centrifuged. The cells were resuspended in mTESR1 (serum-free and feeder-free) medium. After overnight incubation, the cells were reprogrammed for 4–6 weeks using the Simplicon reprogramming kit according to the manufacturing SOP.

[0146] The non-integrated vector used for reprogramming fibroblasts into iPSCs was a modified non-infectious, self-replicating polycistronic Venezuelan encephalitis (VEE) virus RNA replicon RNA system that consistently expressed reprogramming factors across multiple cell divisions. iPSC colonies formed within 4–6 weeks, and the cultures were replated using RelesR (Stem Cell Technologies, enzyme-free reagent) for proliferation. The iPSCs were then passaged weekly for 7–10 passages, at which point they were stabilized (approximately 5–7 weeks). MTesR1 medium containing 1x antibiotics and antifungals was used for the iPSCs, which were filtered and sterilized using a 0.22 μm filter and stored at 4°C for 5–7 days, with daily replacement. Single-cell suspensions were then cryopreserved using ProFreeze CDM according to the manufacturer's protocol. A summary of the results and quality assurance tests is shown in Table 2. [Table 2]

[0147] Stage 3 (From decompression of iPSC bank to RPE bank) RPEs were prepared and stored as described in Hazim et al., Stem Cell Res.Ther. 10(1):52 (2019). Briefly, patient-specific iPSCs were thawed for 1 minute in a temperature-controlled water bath at 37°C. The cells were then centrifuged and cultured in MTesR1 medium, which was changed daily for 5-7 days until the AiPS cells reached 80%-95% confluence.

[0148] For differentiation into RPE cells, the culture medium was switched to a basal medium containing DMEM / F12 supplemented with 14% xeno-free knockout serum substitute (KSR), 1% non-essential amino acids (NEAA), 2 mM L-glutamine, and 10 mM nicotinamide, and the cells were cultured for two weeks. The medium was changed every other day. At weeks 3 and 4 of differentiation, 140 ng / ml of activin A and 20 ng / ml of basic fibroblast growth factor (FGF) were added to the medium, and the cells were grown for a further two weeks. The cell culture was then returned to the basal medium until visual analysis using a phase-contrast microscope showed that most of the cells were deeply pigmented. The pigmented areas began to appear between weeks 5 and 6 of differentiation.

[0149] The pigmented regions were differentially passaged in TrypLE and RPE medium containing DMEM / F12 supplemented with 5% fetal bovine serum (FBS), 4% human AB serum, 0.25 mg / ml taurine, 10 mM nicotinamide, 1×NEAA, 1×N2, 1×B27, 0.1 mM beta-mercaptoethanol, and 1×Glutamax®. The pigmented cells were mechanically passaged by gently collecting the cells in the medium after 10 minutes of TrypLE® treatment. As shown in Table 3, AiPSC-RPE cells passaged with TrypLE for 10 minutes passed identity, safety, stability, purity, and efficacy tests compared to AiPSC-RPE cells passaged with TrypLE for 1 or 3 minutes. [Table 3-1] [Table 3-2] [Table 3-3]

[0150] Next, the cells are cultured at a rate of 10,000 cells / cm² for expansion. 2The cells were replated at a specific density and incubated overnight at 37°C in 5% CO2. RPE growth and purification were continued for approximately 12 weeks, and cell morphology was examined at each passage using a novel morphological scoring system developed to determine the optimal phenotype of the AiPSC-RPE product. The scoring system helped guide critical decisions in the manufacturing process for producing high-quality RPE cells. AiPSC-RPE cells were grown in 6-well plates, and five random images were taken to score the cells based on the morphological scoring system shown in Figure 2. The scoring system was used to determine the number of passages required to culture AiPSC-RPE to obtain a final product that met the identity, safety, purity, potency, and stability criteria. This system eliminated inter-donor variability, resulting in a potent, consistent product. Table 4 below shows two AiPSC-RPE products from passage 3 that did not pass the morphological scoring system. However, after four passages, the cells not only passed the morphological scoring system but also all additional quality criteria. [Table 4-1] [Table 4-2]

[0151] Single-cell suspensions of RPE cells were cryopreserved using ProFreeze CDM according to the manufacturer's protocol. Cell samples were collected for in-process DS release testing, as shown in Table 5. [Table 5-1] [Table 5-2]

[0152] Step 4 - Improved AiPSC-RPE products can be obtained by culturing cells for 4-6 weeks before the final formulation. To understand the optimal conditions for the clinical formulation, the viability and potency of freshly thawed AiPSC-RPE and AiPSC-RPE cultured for 4 weeks were tested. All cells were resuspended in equilibrium salt solution before testing, as intended for surgical delivery. Compared to freshly thawed AiPSC-RPE, AiPSC-RPE cultured for 4 weeks showed a dramatic increase in both cell viability (Figure 3A) and cell potency (Figure 3B), resulting in a significantly more favorable profile for transplantation and leading to its selection as the final formulation.

[0153] AiPSC-RPE pharmaceuticals were tested in equilibrium salt solution formulation buffer, cells were passed through a delivery device, and cells were counted for viability and potency over a continuous period. Cells exhibited significant viability 6 hours after final formulation preparation, and although cell aggregation in salt solution was expected and observed, it did not affect the number of recovered cells or the potency of the final product (Abe et al., Curr. Eye Res. 20(4):268-75 (2000); Lund et al., Cloning Stem Cells 8(3):189-99 (2006)). Furthermore, viability after trypan blue dye exclusion and potency after differential secretion of cytokines VEGF and PEDF remained constant and robust, persisting up to 6 hours after passage through the delivery device. (See Figures 4 and 5. Lonza RPE was used as a primary cell line control). Together, these data demonstrate the unique stability and potency of the cell suspension product.

[0154] Example 2 - Identification of a unique RPE-specific protein marker To demonstrate the purity of the AiPSC-RPE product, a unique panel of RPE-specific markers was developed. Protein expression profiles included positivity for RPE markers PMEL17 and CD140b, and negativity for non-RPE lineage markers, GD2 and CD184. As shown in Figures 6A–6D, the purity of RPE cells, defined by the increasing number of PMEL17(+) / CD140b(+) / GD2(-) / CD184(-) populations, was increased from passage 0 to passage 4 using TrypLE in the step 3 differentiation process described in Example 1.

[0155] Example 3 - Evaluation of the identity, safety, and efficacy of the AiPSC-RPE product from Step 4 To determine variability and consistency between donor cultures, three patient-specific AiPSC-RPE cell products prepared from three parallel cultures from the same RPE bank were tested for identity, safety, and efficacy, as shown in the schematic diagram in Figure 7. Gene expression was evaluated using ddPCR to compare identity. Furthermore, mycoplasma testing was performed to assess safety, and efficacy was evaluated using phagocytic activity tests for each parallel culture.

[0156] The results shown in Table 6 demonstrate that the parallel cultures were equivalent in terms of identity, safety, purity, and potency, and that the parallel culture model is suitable for surrogate release testing of drug products (DPs) administered to patients. [Table 6]

[0157] Example 4 - Evaluation of the efficacy of retinal pigment epithelial cells when administered subretinically to a rat model by the Royal Society of Surgeons (RCS) of England. To determine the efficacy of AiPSC-RPE products in the RCS rat model, AiPSC-RPE products from three independent patient donors (prepared as described in Example 1) and one AiPSC-RPE product from a single patient donor (derived from separate iPSC strains from the single patient donor) were tested in the RCS rat model of retinal degeneration. RCS rats are known to have genetic photoreceptor degeneration (Lund et al., Cloning Stem Cells 8(3):189-99 (2006)).

[0158] method 84 RCS rats (48 males and 36 females) were assigned to the experiment. At postnatal (P)19–23 days, rats were assigned to one of 15 groups according to the study design in Table 7: Groups 1, 6, and 12-AiPSC-RPE cells (01F1i1R1) with a dose of 60,000 cells / eye (treatment); Groups 2, 7, and 13-AiPSC-RPE cells (02F1i1R1) with a dose of 60,000 cells / eye (treatment); Groups 3, 8, and 14-AiPSC-RPE cells (13F1i1R1) with a dose of 60,000 cells / eye (treatment); Groups 4, 9, and 15-AiPSC-RPE cells (13F1i2R1) with a dose of 60,000 cells / eye (treatment); Group 5-vehicle only (control); and Group 10-untreated (control). All animals received immunosuppressive therapy with dexamethasone (daily intraperitoneal infusion for 14 days after administration or assignment to the study) and cyclosporine A (continuous administration via drinking water for the remainder of the study after administration). Clinical ophthalmological examinations were performed at baseline and at weeks 4, 8, and 12 after administration of the test substance and vehicle control. OCT and color fundus photographs were performed on day 0 immediately after administration for animals in groups 1 and 2, and at weeks 4, 8, and 12 for all groups. Animal visual acuity was tested using optokinetic response (OKR), and electroretinography (ERG) was performed at weeks 4, 8, and 12. General health status was observed and recorded daily. Body weight was measured at baseline, week 1, and before the end of the study. Animals were euthanized at weeks 4, 8, or 12. Blood was collected for clinicopathological analysis, and macroscopic autopsies were performed on all animals to collect and analyze the eyes (global), ocular appendages, systemic tissues, and macroscopic lesions. [Table 7]

[0159] result OCT was used to assess ONL thickness at several time points. At 4 weeks post-transplant, ONL increased by more than 15% across all AiPSC-RPE products tested compared to control animals injected with vehicle alone. At 8 weeks post-transplant, an increase of more than 5% in ONL was observed across all AiPSC-RPE products compared to vehicle-only controls (Figure 8, using Dunnett's method for multiple comparisons, corrected p-value < 0.01 for testing all patient-derived cell lines against vehicle alone). As photoreceptor degradation in the ONL of RCS rats progressed, the increase in ONL ceased, and the results became inconclusive.

[0160] Furthermore, the presence of transplanted cells in the subretinal region was investigated using human markers. The persistence of AiPSC-RPE cells at all time points measured from week 4 to week 12 post-transplant (Table 8) demonstrated the robustness of the AiPSC-RPE cell product. [Table 8]

[0161] Immunocytochemistry showed human nuclear protein expression at 4 and 12 weeks post-transplant (Figures 9 and 10, respectively). Results at 8 weeks were consistent (data not shown). Importantly, at 12 weeks, there was evidence that AiPSC-RPE cells had migrated from the injection site (Figure 11, area enclosed in white) and incorporated into a monolayer with an endogenous RPE layer. The cells also showed polarization, as evident by ezrin staining at the apical end (data not shown).

[0162] As an in vivo functional test, OKRs were performed on the animal cohort up to 12 weeks post-transplant. The OKR response in 01F1i1R1 showed an increase of over 7% at 8 weeks and a 4% increase at 12 weeks. The OKR response in 02F1i1R1 showed a 3% increase at both 8 and 12 weeks post-transplant. The greatest increases in OKR response were observed in the AiPSC-RPE cell products 13F1i1R1 and 13F1i2R1, showing an increase of over 7% compared to the vehicle control-only group throughout the entire trial from 4 to 12 weeks (Figure 12, using Dunnett's method for multiple comparisons, adjusted p-value < 0.01 for all patient-derived cell lines compared to vehicle alone).

[0163] Furthermore, retinal function was tested using ERG. At week 4, dark adaptation b-wave amplitude increased with increasing flash intensity for all treatment groups to 39.8 cd·sec / m². 2 At maximum intensity, the dark-adapted b-wave amplitude was higher in eyes treated with 01F1i1R1 or 02F1i1R1 compared to vehicle-treated and untreated eyes. The amplitude was 39.8 cd·sec / m² in eyes treated with 13F1i1R1 or 13F1i2R1. 2 The dark-adapted b-wave amplitude at this point was comparable between the vehicle-treated eye and the uninjected eye. By week 8, the dark-adapted b-wave amplitude still increased with increasing flash intensity, but to a much smaller extent than at week 4. 39.8 cd·sec / m 2 At maximum intensity, the dark-adapted b-wave amplitude in eyes treated with 01F1i1R1 or 02F1i1R1 decreased compared to week 4. However, in eyes treated with 02F1i1R1, the dark-adapted b-wave amplitude was still slightly higher than in vehicle-treated and untreated eyes. The mean dark-adapted b-wave amplitude measured for eyes treated with 13F1i1R1 or 13F1i2R1 was lower than in eyes injected with other cells, but slightly lower than in vehicle-treated and untreated eyes. By week 12, the dark-adapted b-wave amplitude no longer consistently increased with increasing flash intensity. 39.8 cd·sec / m 2At the highest intensity, the dark adaptation b-wave amplitude in vehicle-treated and untreated eyes decreased sharply compared to week 8, and the mean dark adaptation b-wave amplitude in eyes treated with 01F1i1R1 and 13F1i2R1 was higher than in vehicle-treated and untreated eyes (Figure 13).

[0164] conclusion Subretinal injection of 60,000 postmittal AiPSC-RPE cells per eye from one of four different cell lines into the eyes of immunosuppressed RCS rats was associated with the formation of AiPSC-RPE cell grafts in the subretinal space, and increased retinal ONL thickness at 4 and 8 weeks post-AiPSC-RPE cell transplantation (approximately ages P49 and P77, respectively) compared to vehicles-injected and untreated eyes. Pathological signs of retinal degeneration, including retinal autofluorescence, retinal vascularization, RPE aggregation, and optic disc pallor, were also delayed and / or reduced in severity at these time points in AiPSC-RPE-injected eyes compared to vehicles-injected and untreated eyes. These findings suggest that treatment with AiPSC-RPE cells ameliorated the photoreceptor degeneration and retinal deterioration characteristic of RCS rats. Despite the rapid deterioration of the retinas in RCS rats, functionally positive results were found at multiple time points, indicating a potential therapeutic effect. Therefore, ERG analysis and OKR evaluation suggested the potential for improved retinal function in dystrophy RCS rats after RPE cell transplantation, as evidenced by enhancement of ERG b waves and higher OKR gains.

[0165] The persistence and function of AiPSC-RPE cells were tested in a rat model of retinal degeneration with RCS, a reasonably characterized animal model for RPE dysfunction in vivo. Both robust persistence and appropriate function of AiPSC-RPE DP were observed.

[0166] Multiple safety, toxicity, and tumorigenicity studies were conducted in SCID beige mice and RNU rats (immunodeficient animals), and no adverse effects of the AiPSC-RPE product were observed up to 3 months post-transplantation, both in terms of administration route and testes (a highly tolerable tissue for tumor measurement).

[0167] Other Embodiments While several embodiments of the present invention are described herein, this disclosure and examples may be modified to provide other methods and compositions of the present invention. It will be understood that the scope of the present invention should be defined by the appended claims, in addition to the specific embodiments shown as examples. All references cited herein are incorporated herein by reference.

Claims

1. A preparation comprising retinal pigment epithelial (RPE) cells differentiated from induced pluripotent stem cells (iPSCs), wherein at least 80% of the RPE cells are PMEL17(+) / CD140b(+) / GD2(-) / CD184(-).

2. The preparation according to claim 1, wherein at least 80% of the RPE cells express RPE65, BEST1, RLBP1, and MerTK.

3. The preparation according to claim 1 or 2, wherein, when detected by digital droplet PCR (ddPCR), less than 0.002% of the RPE cells are positive for Lin28 and do not express Oct4 or Klf4.

4. The preparation according to any one of claims 1 to 3, wherein the culture of the preparation comprises RPE cells in which at least 95% of the RPE cells exhibit confluent hexagonal cells, pigmentation, cobblestone structure, well-defined membrane boundaries, and polarization (e.g., as evaluated using ezrin staining).

5. (a) The RPE cells exhibit a transepithelial electrical resistance (TEER) of at least 100 ohms / cm² after being cultured for about 1 to 8 weeks. (b) The RPE cells exhibit differential secretion of VEGF at a ratio of basal secretion to apical secretion greater than 0.5, (c) The RPE cells exhibit differential secretion of PEDF at a ratio of apical secretion to basal secretion greater than 0.5, and (d) At least 50% of the RPE cells exhibit the ability to phagocytose photoreceptor outer segments, as measured, for example, by flow cytometry. The preparation according to any one of claims 1 to 4.

6. The preparation according to any one of claims 1 to 5, wherein at least 80% of the RPE cells are viable when passed through an implantation device (e.g., a 41g device), as determined by, for example, trypan blue staining.

7. The preparation according to any one of claims 1 to 6, wherein, during passaging via an implantation device (e.g., a 41g device), at least 80% of the RPE cells exhibit a TER of at least about 50 ohms / cm², at least about 75 ohms / cm², at least about 100 ohms / cm², at least about 125 ohms / cm², at least about 150 ohms / cm², at least about 175 ohms / cm², or at least about 200 ohms / cm², for example, after 1 to 8 weeks of culture.

8. The preparation according to claim 7, wherein at least 80% of the RPE cells are viable after storage at room temperature for 6 hours, as determined, for example, by trypan blue staining.

9. A preparation comprising retinal pigment epithelial (RPE) cells differentiated from induced pluripotent stem cells (iPSCs), wherein the preparation has undergone at least one freeze / thaw cycle and has a viability of at least 80%, 90%, or 95% (as determined, for example, by trypan blue staining), compared to a reference preparation comprising RPE cells differentiated from iPSCs and having undergone at least one freeze / thaw cycle.

10. The preparation according to claim 9, wherein at least 80%, 90%, or 95% of the RPE cells of the preparation are viable after at least one freeze / thaw cycle of the preparation (as determined, for example, by trypan blue staining).

11. The preparation according to claim 9 or 10, wherein after at least one freeze / thaw cycle, the preparation is cultured in a culture medium for about two weeks to about six weeks, for example, about four weeks.

12. The preparation according to any one of claims 9 to 11, wherein at least 80% of the RPE cells are PMEL17(+) / CD140b(+) / GD2(-) / CD184(-).

13. The preparation according to any one of claims 9 to 12, wherein at least 80% of the RPE cells express RPE65, BEST1, RLBP1, and MerTK.

14. The preparation according to any one of claims 9 to 13, wherein, when detected by digital droplet PCR (ddPCR), less than 0.002% of the RPE cells are positive for Lin28 and do not express Oct4 or Klf4.

15. The preparation according to any one of claims 9 to 14, wherein the culture of the preparation comprises RPE cells in which at least 95% of the RPE cells exhibit confluent hexagonal cells, pigmentation, cobblestone structure, well-defined membrane boundaries, and polarization (e.g., as evaluated using ezrin staining).

16. (a) The RPE cells exhibit a transepithelial electrical resistance (TEER) of at least 100 ohms / cm² after being cultured for about 1 to 8 weeks. (b) The RPE cells exhibit differential secretion of VEGF at a ratio of basal secretion to apical secretion greater than 0.5, (c) The RPE cells exhibit differential secretion of PEDF at a ratio of apical secretion to basal secretion greater than 0.5, and (d) At least 50% of the RPE cells exhibit the ability to phagocytose photoreceptor outer segments, as measured, for example, by flow cytometry. The preparation according to any one of claims 9 to 15.

17. The preparation according to any one of claims 9 to 16, wherein at least 80% of the RPE cells are viable when passed through an implantation device (e.g., a 41g device), as determined by, for example, trypan blue staining.

18. The preparation according to claim 17, wherein at least 80% of the RPE cells are viable after storage at room temperature for 6 hours, as determined, for example, by trypan blue staining.

19. The preparation according to any one of claims 1 to 18, wherein the iPSC is derived from fibroblasts or peripheral blood mononuclear cells.

20. The preparation according to any one of claims 1 to 19, comprising the suspension of RPE cells.

21. The preparation according to any one of claims 1 to 19, comprising a scaffold, matrix, or bioink, wherein the RPE cells are seeded, cultured, printed, or embedded on the scaffold, matrix, or bioink.

22. A preparation according to any one of claims 1 to 21, comprising approximately 10,000 to approximately 1,500,000 RPE cells.

23. A method for producing a preparation of RPE cells, wherein the method is A step of growing fibroblasts from a skin sample, or a step of growing peripheral blood mononuclear cells from a whole blood sample; A step of producing iPSCs from the fibroblasts or peripheral blood mononuclear cells; The step of differentiating the iPSC into a culture of RPE cells; and The step of subculturing the cultured RPE cells with a dissociation reagent for at least 10 minutes until at least 95% of the cultured RPE cells exhibit confluent hexagonal cells, pigmentation, cobblestone morphology, and well-defined membrane boundaries, thereby producing a preparation of RPE cells. Methods that include...

24. The method according to claim 23, further comprising the step of subjecting the preparation to at least one freeze / thaw cycle.

25. The method according to claim 23 or 24, wherein at least 80% of the RPE cells in the preparation are PMEL17(+) / CD140b(+) / GD2(-) / CD184(-).

26. The method according to any one of claims 23 to 25, wherein at least 80% of the RPE cells of the preparation express RPE65, BEST1, RLBP1, and MerTK.

27. For example, the method according to any one of claims 23 to 26, wherein, when detected by digital droplet PCR (ddPCR), less than 0.002% of the RPE cells of the preparation are positive for Lin28 and do not express Oct4 or Klf4.

28. (a) The RPE cells exhibit a transepithelial electrical resistance (TEER) of at least 100 ohms / cm² after being cultured for about 1 to 8 weeks. (b) The RPE cells exhibit differential secretion of VEGF at a ratio of basal secretion to apical secretion greater than 0.5, (c) The RPE cells exhibit differential secretion of PEDF at a ratio of apical secretion to basal secretion greater than 0.5, and (d) At least 50% of the RPE cells exhibit the ability to phagocytose photoreceptor outer segments, as measured, for example, by flow cytometry. The method according to any one of claims 23 to 27.

29. The method according to any one of claims 23 to 28, wherein at least 80% of the RPE cells are viable when passed through a transplantation device (e.g., a 41g device), as determined, for example, by trypan blue staining.

30. The method according to claim 29, wherein at least 80% of the RPE cells are viable after storage at room temperature for 6 hours, as determined, for example, by trypan blue staining.

31. A preparation of RPE cells prepared by the method described in any one of claims 23 to 30.

32. A method for treating a subject who is suffering from or at risk of an eye disorder, comprising the step of administering a preparation of RPE cells according to any one of claims 1 to 22 or 31 to the eye of the subject.

33. The method according to claim 32, wherein the RPE cells are self-referential to the subject.

34. The method according to claim 32 or 33, wherein the preparation contains about 10,000 to about 1,500,000 RPE cells.

35. The method according to any one of claims 32 to 34, wherein the preparation is introduced into any region of the eye of the subject.

36. The method according to any one of claims 32 to 35, wherein the preparation comprises the suspension of RPE cells.

37. The method according to any one of claims 32 to 35, wherein the preparation comprises a scaffold, a matrix, or a bioink, and the RPE cells are seeded, cultured, printed, or embedded on the scaffold, matrix, or bioink.

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