Methods for measuring therapeutic effects of retinal disease therapies

Abstract

To provide compositions and methods for treating retinal diseases or disorders using RPE cells.SOLUTION: The present invention provides a pharmaceutical composition for treating or slowing the progression of a retinal disease or disorder in a subject, comprising a therapeutically effective amount of retinal pigment epithelium (RPE) cells. The therapeutically effective amount of RPE cells is about 50,000 and 5,000,000 cells. The composition is formulated for implantation to a region covering the geographic atrophy (GA) lesion or a region covering the fovea in a subject.SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to and benefit of U.S. Provisional Patent Application No. 62 / 501,690, filed November 13, 2017, and U.S. Provisional Patent Application No. 62 / 472,544, filed March 16, 2017, and U.S. Provisional Patent Application No. 62 / 585,520, filed May 4, 2017, each of which is incorporated herein by reference in its entirety.

[0002] The present disclosure relates generally to the field of treating retinal diseases, and more particularly to treating retinal diseases using human embryonic stem cell-derived retinal pigment epithelial (RPE) cell compositions. [Background technology]

[0003] Dysfunction, degeneration, and loss of RPE cells are hallmarks of retinal diseases, including AMD, Best's disease, and subtypes of retinitis pigmentosa (RP). AMD is the leading cause of visual impairment in the Western world. Among people over the age of 75, 25–30% suffer from age-related macular degeneration (AMD), with progressive central vision loss leading to blindness in 6–8% of patients. Retinal degeneration primarily involves the macula, the central part of the retina responsible for fine visual detail and color perception, face recognition, reading, and driving. Dry AMD is initiated by RPE hyperplasia and the formation of drusen deposits beneath the RPE or within Bruch's membrane, which consists of metabolic end products. This disease gradually progresses to the advanced stage of geographic atrophy (GA), which involves degeneration of RPE cells and photoreceptors over large areas of the macula and can cause central vision loss.

[0004] The pathogenesis of this disease involves abnormalities in four functionally interrelated tissues: the retinal pigment epithelium (RPE), Bruch's membrane, the choriocapillaris, and the photoreceptors. However, impaired RPE cell function is an early and critical event in the molecular pathway that leads to clinically relevant AMD changes.

[0005] Currently, there are no effective or approved treatments for dry AMD. Preventive measures include vitamin / mineral supplements, which reduce the risk of developing wet AMD but do not affect the development of geographic atrophy progression.

[0006] Because best-corrected visual acuity (BCVA) is a measure of foveal visual acuity, if the fovea is intact, BCVA scores may not be affected. BCVA is widely accepted by clinical communities and regulatory authorities worldwide as an important measure of visual function and is the gold standard by which the effectiveness of retinal disease treatments is judged. However, it may sometimes fail to assess the nuances of comprehensive visual function. Evidence has shown that in subjects with a BCVA of 20 / 50 or better, other features of visual function, including contrast sensitivity, low-luminance BCVA, and reading speed, can be significantly impaired. Furthermore, best-corrected visual acuity alone does not adequately measure the progression of visual impairment in all subjects, including those with foveal-avoidance GA. Summary of the Invention

[0007] overview Retinal pigment epithelial (RPE) cells and RPE cell compositions have been developed that are useful for treating retinal diseases and disorders, including preventing the progression of retinal degeneration and blindness. These RPE cells and cell compositions safely promote the engraftment, integration, survival, and function of ocular structures when administered to a subject in need thereof.

[0008] Visual impairment, retinal disease progression, and the effectiveness of retinal disease treatments can be detected and monitored using quantitative morphological assessment techniques, even in subjects with intact BCVA. Clinical trials involving subjects with AMD and GA aimed at quantifying changes in visual function and correlating them with disease progression can incorporate additional assessments that account for the underlying pathophysiological processes of the disease. Also disclosed herein are methods for measuring the therapeutic effects of retinal disease treatments using improved quantitative structural and functional assessments.

[0009] According to some aspects, provided herein are methods of treating or slowing the progression of a retinal disease or disorder, the methods comprising administering to a subject a therapeutically effective amount of a pharmaceutical composition comprising retinal pigment epithelial (RPE) cells.

[0010] In some embodiments, administration of a therapeutically effective amount of retinal pigment epithelial (RPE) cells results in best corrected visual acuity (BCVA) that remains unchanged for about 1 day to about 3 months, 1 day to about 15 months, or 1 day to about 24 months, or about 90 days to about 24 months, measured from baseline.

[0011] In some embodiments, the subject has a BCVA of 20 / 64 or less, 20 / 70 or less, or from about 20 / 64 to about 20 / 400.

[0012] In some embodiments, administration of a therapeutically effective amount of retinal pigment epithelial (RPE) cells results in a best corrected visual acuity (BCVA) that remains stable for about 1 day to about 15 months, or 1 day to about 24 months, or about 90 days to about 24 months, as measured from baseline.

[0013] In some embodiments, administration of a therapeutically effective amount of retinal pigment epithelial (RPE) cells results in increased pigmentation in about 89% to about 96% of subjects. In other embodiments, the increased pigmentation is maintained for at least about 6 months to about 12 months, or for about 90 days to about 24 months. In still other embodiments, administration of a therapeutically effective amount of retinal pigment epithelial (RPE) cells results in retinal pigmentation.

[0014] In further embodiments, administration of a therapeutically effective amount of retinal pigment epithelial (RPE) cells results in an increase in retinal pigmentation measured from baseline for at least about 2 months to about 1 year, or 90 days to about 24 months. In other embodiments, retinal pigmentation stabilizes at about 2 to about 12 months, or about 90 days to about 24 months after administration. In yet other embodiments, retinal pigmentation stabilizes at about 3 to about 9 months after administration.

[0015] According to some aspects of the present disclosure, the subretinal fluid within the bleb into which the cells are administered is absorbed in less than 48 hours.

[0016] According to another aspect, administration of a therapeutically effective amount of retinal pigment epithelial (RPE) cells results in restoration of the ellipsoid area. In yet another aspect, restoration of the ellipsoid area comprises restoration by ellipsoid area analysis.

[0017] In some embodiments, the ellipsoid area analysis involves visual analysis of the ellipsoid area and comparing the subject's ellipsoid area to that of an age-matched and gender-matched control, baseline, or fellow eye.

[0018] According to further embodiments, recovery is indicated by restoration of normal structure compared to an age-matched and gender-matched control, baseline, or fellow eye. According to other embodiments, recovery includes a subjective assessment that one or more of the following are becoming more organized: the outer limiting membrane, the myoid zone (inner photoreceptor segment), the ellipsoid zone (IS / OS junction), the outer photoreceptor segment, loss of drusen, and disappearance of retinal pseudodrusen. In some embodiments, recovery includes a subjective assessment that one or more of the basic underlying layers of the retina are becoming more organized.

[0019] According to certain embodiments, the basic underlying layers of the becoming more organized retina include one or more of the outer limiting membrane, the myoid region (inner segments of photoreceptors), the ellipsoid region (IS / OS junction), and the outer segments of photoreceptors.

[0020] According to other embodiments, the new or worsening ERM does not require surgical removal within about 1 week to about 12 months, or about 1 week to about 24 months, or about 90 days to about 24 months of administration.

[0021] According to some embodiments, the RPE cells do not exhibit tumorigenicity within about 1 week to about 1 year, or about 1 week to about 24 months, or about 90 days to about 24 months of administration.

[0022] According to some embodiments, the RPE cells exhibit a histological tumorigenicity of 0% to about 5% within about 9 months of administration.

[0023] According to some embodiments, administration of a therapeutically effective amount of retinal pigment epithelial (RPE) cells does not result in retinal tears or ruptures.

[0024] According to some embodiments, administration of a therapeutically effective amount of retinal pigment epithelial (RPE) cells does not result in retinal edema.

[0025] According to some embodiments, the therapeutically effective amount of RPE cells is between about 50,000 and 5,000,000 cells per administration.

[0026] According to some embodiments, the therapeutically effective amount of RPE cells is about 200,000 cells per administration.

[0027] According to some embodiments, the therapeutically effective amount of RPE cells is about 500,000 cells per administration.

[0028] According to some embodiments, the pharmaceutical composition comprises about 500 cells / μl to about 10,000 cells / μl.

[0029] According to some embodiments, when the amount is 50,000 cells per dose, the pharmaceutical composition contains about 500-1,000 cells / μl.

[0030] According to some embodiments, when the amount is 200,000 cells per dose, the pharmaceutical composition contains about 2,000 cells / μl.

[0031] According to some embodiments, when the amount is 500,000 cells per dose, the pharmaceutical composition contains about 5,000 cells / μl.

[0032] According to some embodiments, when the amount is 1,000,000 cells per dose, the pharmaceutical composition contains about 10,000 cells / μl.

[0033] According to some embodiments, at least 95% of the cells co-express the pre-melanosome protein (PMEL17) and the cellular retinaldehyde-binding protein (CRALBP).

[0034] According to some embodiments, the transepithelial electrical resistance of the cells is greater than 100 ohms relative to the subject.

[0035] According to some embodiments, the RPE cells are generated by ex vivo differentiation of human embryonic stem cells.

[0036] According to some embodiments, the administering comprises transplanting RPE cells.

[0037] According to some embodiments, the methods described herein further comprise preparing an RPE dose prior to RPE cell transplantation. According to some embodiments, preparing the RPE dose comprises thawing the dose. According to some embodiments, preparing the RPE dose comprises mixing the RPE cells and loading them into a delivery device.

[0038] According to some embodiments, the methods described herein further comprise performing a vitrectomy prior to RPE cell transplantation. According to some embodiments, performing a vitrectomy comprises administering triamcinolone to stain the vitreous and removing vitreous traction.

[0039] According to certain aspects, the methods described herein further comprise cleansing the surgical site prior to performing the vitrectomy.

[0040] According to some embodiments, the methods described herein further comprise irrigating the surgical site after transplanting the RPE cells.

[0041] According to some embodiments, administration includes cleaning the surgical site, performing the vitrectomy, preparing the RPE dose, and transplanting the RPE cells.

[0042] According to some embodiments, transplanting RPE cells comprises injecting the RPE cells at least one papillary diameter away from the edge of the geographic atrophy (GA) lesion.

[0043] According to some embodiments, transplantation of RPE cells includes injecting RPE cells in one or more of the following steps: covering the GA lesion, covering the fovea, covering part or all of the transition zone bordering the GA lesion, or covering surrounding healthy tissue adjacent to the GA lesion.

[0044] According to some embodiments, the transition zone comprises the area between the intact retina and the degenerated retina.

[0045] According to some embodiments, covering the GA lesion includes covering the entire GA lesion with a bleb. According to other embodiments, the size of the GA is less than 0.1 mm. 2~ Approximately 50 mm 2 , about 0.5mm 2 ~about 30mm 2 , about 0.5mm 2 ~about 15mm 2 , about 0.1mm 2 ~about 10mm 2 , about 0.25mm 2 ~about 5mm 2 , or any point between the two points.

[0046] According to some embodiments, administering comprises administering RPE cells such that central macular vision is preserved.

[0047] According to some embodiments, RPE cells are produced by (a) culturing human embryonic stem cells or induced pluripotent stem cells in a medium containing nicotinamide to produce differentiated cells; (b) culturing the differentiated cells in a medium containing nicotinamide and activin A to produce cells that further differentiate into the RPE lineage; and (c) culturing the cells that further differentiate into the RPE lineage in a medium containing nicotinamide but not activin A.

[0048] According to some embodiments, the embryonic stem cells or induced pluripotent stem cells are grown under non-adherent conditions in a medium comprising bFGF and TGFβ. According to further embodiments, the medium of (a) is substantially free of activin A.

[0049] In some embodiments, the cells are administered in a single dose. In some embodiments, the cells are administered to the subretinal space of the subject. In some embodiments, the subretinal administration is intravitreal or suprachoroidal. In some embodiments, the administration is via a cannula.

[0050] According to some embodiments, the cannula administration site heals within about 1 to about 30 days, and according to some embodiments, the cannula administration site heals within about 5 to about 21 days or about 7 to about 15 days.

[0051] According to some embodiments, the methods described herein further comprise administering immunosuppression to the subject for 1 day to 3 months following administration of the RPE cells.

[0052] According to other embodiments, the methods described herein further comprise administering immunosuppression to the subject for three months following administration of the RPE cells.

[0053] According to yet another embodiment, the methods described herein further comprise administering immunosuppression to the subject for 1 day to 1 month following administration of the RPE cells.

[0054] According to some embodiments, the retinal disease or condition is selected from the group consisting of moderate atrophic AMD, retinitis pigmentosa, retinal detachment, retinal dysplasia, retinal atrophy, retinopathy, macular dystrophy, cone dystrophy, cone-rod dystrophy, Malattia Leventinese, Doyne honeycomb dystrophy, Sorsby's dystrophy, pattern / sphenoid dystrophy, Best vitelliform dystrophy, North Carolina dystrophy, central areolar choroidal dystrophy, angioid streaks, toxic maculopathy, Stargardt's disease, pathological myopia, retinitis pigmentosa, and macular degeneration.

[0055] In some embodiments, the disease is age-related macular degeneration. In some embodiments, the age-related macular degeneration is dry age-related macular degeneration.

[0056] According to some aspects, provided herein are methods of enhancing the safety of a method of treating a subject with dry AMD, comprising administering a therapeutically effective amount of retinal pigment epithelial (RPE) cells to a subject not receiving systemic immunosuppression.

[0057] According to some embodiments, the incidence and frequency of treatment-emergent adverse events is lower than with immunosuppression.

[0058] According to some aspects, provided herein is a method for organizing ellipsoid regions of the retina in a subject with GA, the method comprising administering a therapeutically effective amount of retinal pigment epithelial (RPE) cells, wherein after administration, the disorganized ellipsoid regions become organized.

[0059] According to some embodiments, recovering the ellipsoidal region comprises recovering by ellipsoidal region analysis.

[0060] According to some embodiments, the ellipsoid area analysis involves visual analysis of the ellipsoid area and comparing the subject's ellipsoid area to an age-matched and gender-matched control, baseline, or fellow eye.

[0061] According to some embodiments, recovery is indicated by restoration of normal structure compared to an age-matched and gender-matched control, baseline, or fellow eye.

[0062] According to some embodiments, recovery includes a subjective assessment that one or more of the external limiting membrane, myoid region (inner photoreceptor segments), ellipsoid region (IS / OS junction), outer photoreceptor segments, loss of drusen, and disappearance of reticular pseudodrusen are becoming more organized.

[0063] According to some embodiments, recovery comprises a subjective assessment that one or more of the basic underlying layers of the retina are becoming more organized.

[0064] According to some embodiments, the basic underlying layers of the retina that are becoming more organized include one or more of the outer limiting membrane, the myoid region (inner segments of photoreceptors), the ellipsoid region (IS / OS junction), and the outer segments of photoreceptors.

[0065] According to some embodiments, the subject has a BCVA of 20 / 64 or less, 20 / 70 or less, or from about 20 / 64 to about 20 / 400.

[0066] According to some embodiments, treating or slowing the progression of retinal disease is demonstrated by recovery of visual acuity as assessed by microperimetry, where recovery of visual acuity as assessed by microperimetry comprises a correlation between retinal sensitivity and EZ loss in microperimetry compared to baseline.

[0067] According to other embodiments, recovery of visual acuity as assessed by microperimetry includes demonstrating that an area of ​​the retina at or near the site of administration of the RPE cells comprises an improved microperimetry assessment compared to a baseline microperimetry assessment.

[0068] According to certain embodiments, treating or slowing the progression of retinal disease comprises a reduction in the rate of GA lesion growth by about 5% to about 20%, or about 5% to about 50%, or about 5% to about 25%, or about 5% to about 100%, or about 5% to about 10%, compared to baseline or the fellow eye at one year after administration.

[0069] According to some embodiments, treating or slowing the progression of retinal disease includes one or more of the following: stable BCVA when compared to an age-matched and gender-matched control, baseline, or fellow eye; no decline in low-light test performance; or no decline in sensitivity in microperimetry; or no decline in reading speed, wherein the comparison is at one or more of 1 month, 3 months, 6 months, or 1 year.

[0070] According to some embodiments, there is provided a pharmaceutical composition for treating or slowing the progression of a retinal disease or disorder, comprising approximately 50,000 to 500,000 RPE cells as an active agent.

[0071] According to another aspect, there is provided a pharmaceutical composition for stabilizing the RPE in a subject with a retinal disease or disorder, comprising approximately 50,000 to 500,000 RPE cells as an active agent.

[0072] According to some embodiments, the RPE cells are characterized by the following properties: (a) at least 95% of cells co-express a pre-melanosome protein (PMEL17) and a cellular retinaldehyde-binding protein (CRALBP); and (b) the transepithelial electrical resistance of the cells is greater than 100 ohms in a subject to whom the cells are administered, and retinal pigmentation in the subject is stabilized between about 90 days and about 24 months after administration.

[0073] According to some embodiments, restoration of the ellipsoidal region comprises an improvement in one or more of the thickness, area, or volume measurements of the EZ-RPE.

[0074] According to some embodiments, improvement in one or more of the thickness, area, or volume measurements of the EZ-RPE is inversely correlated with visual acuity.

[0075] According to some embodiments, ellipsoidal area analysis demonstrates EZ organization with reduced EZ volume compared to age-matched and gender-matched controls, baseline, or fellow eyes.

[0076] According to some embodiments, the reduction in EZ volume comprises at least 2%, or at least 5%, or at least 7%, or at least 10%, or 1-5%, or 1-10%, or 1-50%, or 10-50%.

[0077] According to some embodiments, organization of the EZ comprises a decrease in the volume of structures in the EZ of at least 2%, at least 5%, at least 10%, about 1% to about 50% from baseline.

[0078] According to some embodiments, treating or slowing the progression of a retinal disease or disorder is enhanced by cellular secretion of trophic factors. [The present invention 1001] A method for treating or slowing the progression of a retinal disease or disorder, comprising administering to a subject a therapeutically effective amount of a pharmaceutical composition comprising retinal pigment epithelial (RPE) cells. [The present invention 1002] The method of the present invention 1001, wherein administration of a therapeutically effective amount of retinal pigment epithelial (RPE) cells results in best corrected visual acuity (BCVA) that does not decline for about 1 day to about 3 months, 1 day to about 15 months, or 1 day to about 24 months, or about 90 days to about 24 months, as measured from baseline. [The present invention 1003] The method of claim 1001, wherein the subject has a BCVA of 20 / 64 or less, 20 / 70 or less, or about 20 / 64 to about 20 / 400. [The present invention 1004] The method of the present invention 1001, wherein administration of a therapeutically effective amount of retinal pigment epithelial (RPE) cells results in a best corrected visual acuity (BCVA) that remains stable for about 1 day to about 15 months, or 1 day to about 24 months, or about 90 days to about 24 months, as measured from baseline. [The present invention 1005] 1001. The method of claim 1001, wherein administration of a therapeutically effective amount of retinal pigment epithelial (RPE) cells results in increased pigmentation in about 89% to about 96% of subjects. [The present invention 1006] The method of claim 1005, wherein the increased pigmentation is maintained for at least about 6 months to about 12 months, or for about 90 days to about 24 months. [The present invention 1007] 1001. The method of claim 1001, wherein administration of a therapeutically effective amount of retinal pigment epithelial (RPE) cells results in retinal pigmentation. [The present invention 1008] 1007. The method of the present invention, wherein administration of a therapeutically effective amount of retinal pigment epithelial (RPE) cells results in an increase in retinal pigmentation measured from baseline for at least about 2 months to about 1 year, or for 90 days to about 24 months. [The present invention 1009] The method of the present invention 1007, wherein retinal pigmentation is stabilized about 2 to about 12 months, or about 90 days to about 24 months after administration. [The present invention 1010] The method of the present invention 1007, wherein retinal pigmentation is stabilized about 3 to about 9 months after administration. [The present invention 1011] 1001. The method of claim 1001, wherein the subretinal fluid in the bleb into which the cells are administered is absorbed in less than 48 hours. [The present invention 1012] 1001. The method of claim 1001, wherein administration of a therapeutically effective amount of retinal pigment epithelial (RPE) cells results in restoration of the ellipsoid area. [The present invention 1013] The method of claim 1012, wherein recovering the ellipsoidal region comprises recovering by ellipsoidal region analysis. [The present invention 1014] The method of claim 1012, wherein the ellipsoid area analysis includes visual analysis of the ellipsoid area and comparing the subject's ellipsoid area to that of an age-matched and gender-matched control, baseline, or fellow eye. [The present invention 1015] The method of claim 1012, wherein recovery is indicated by restoration of normal structure compared to an age-matched and gender-matched control, baseline, or fellow eye. [The present invention 1016] The method of the present invention 1012, wherein recovery includes a subjective assessment that one or more of the external limiting membrane, myoid region (inner segments of photoreceptors), ellipsoid region (IS / OS junction), outer segments of photoreceptors, loss of drusen, and disappearance of reticular pseudodrusen are becoming more organized. [The present invention 1017] The method of claim 1012, wherein recovery comprises a subjective assessment that one or more of the fundamental underlying layers of the retina are becoming more organized. [The present invention 1018] The method of the present invention 1017, wherein the basic underlying layers of the retina that are becoming more organized include one or more of the outer limiting membrane, the myoid region (inner segments of photoreceptors), the ellipsoid region (IS / OS junction), and the outer segments of photoreceptors. [The present invention 1019] The method of the present invention 1001, wherein the new or worsening ERM does not require surgical removal within about 1 week to about 12 months, or about 1 week to about 24 months, or about 90 days to about 24 months of administration. [The present invention 1020] 1001. The method of the present invention, wherein the RPE cells do not exhibit tumorigenicity within about 1 week to about 1 year, or about 1 week to about 24 months, or about 90 days to about 24 months after administration. [The present invention 1021] 1001. The method of claim 1001, wherein the RPE cells exhibit a histological tumorigenicity of 0% to about 5% within about 9 months of administration. [The present invention 1022] 1001. The method of claim 1001, wherein administration of a therapeutically effective amount of retinal pigment epithelial (RPE) cells does not result in retinal tears or ruptures. [The present invention 1023] 1001. The method of claim 1001, wherein administration of a therapeutically effective amount of retinal pigment epithelial (RPE) cells does not result in retinal edema. [The present invention 1024] 1001. The method of claim 1001, wherein the therapeutically effective amount of RPE cells is about 50,000 to 5,000,000 cells per administration. [The present invention 1025] 1001. The method of claim 1001, wherein the therapeutically effective amount of RPE cells is about 200,000 cells per administration. [The present invention 1026] 1001. The method of claim 1001, wherein the therapeutically effective amount of RPE cells is about 500,000 cells per administration. [The present invention 1027] 1001. The method of claim 1001, wherein the pharmaceutical composition comprises about 500 cells / μl to about 10,000 cells / μl. [The present invention 1028] 1001. The method of claim 1001, wherein the pharmaceutical composition contains about 500-1,000 cells / μl when said amount is 50,000 cells per administration. [The present invention 1029] 1001. The method of claim 1001, wherein the pharmaceutical composition contains about 2,000 cells / μl when said amount is 200,000 cells per dose. [The present invention 1030] 1001. The method of claim 1001, wherein the pharmaceutical composition contains about 5,000 cells / μl when said amount is 500,000 cells per dose. [The present invention 1031] 1001. The method of claim 1001, wherein the pharmaceutical composition contains about 10,000 cells / μl when said amount is 1,000,000 cells per dose. [The present invention 1032] 1001. The method of claim 1001, wherein at least 95% of said cells co-express a pre-melanosome protein (PMEL17) and a cellular retinaldehyde-binding protein (CRALBP). [The present invention 1033] The method of claim 1032, wherein the transepithelial electrical resistance of said cells is greater than 100 ohms relative to the subject. [The present invention 1034] 1001. The method of claim 1001, wherein the RPE cells are generated by ex vivo differentiation of human embryonic stem cells. [This invention 1035] 1001. The method of claim 1001, wherein the administering comprises transplanting RPE cells. [The present invention 1036] The method of claim 1035, further comprising preparation of the RPE dose prior to RPE cell transplantation. [This invention 1037] The method of claim 1036, wherein preparing the dose of RPE comprises thawing the dose. [The present invention 1038] The method of claim 1037, wherein preparing the dose of RPE comprises mixing RPE cells and loading them into a delivery device. [This invention 1039] The method of claim 1035, further comprising performing a vitrectomy prior to RPE cell transplantation. [The present invention 1040] The method of claim 1039, wherein performing a vitrectomy comprises administering triamcinolone to stain the vitreous and removing vitreous traction. [The present invention 1041] The method of claim 1035, further comprising cleansing the surgical site before performing the vitrectomy. [The present invention 1042] The method of claim 1035, further comprising irrigating the surgical site after transplanting the RPE cells. [This invention 1043] The method of claim 1001, wherein the administration includes cleaning the surgical site, performing a vitrectomy, preparing an RPE dose, and transplanting RPE cells. [The present invention 1044] 1001. The method of claim 1001, wherein the transplantation of RPE cells comprises injecting RPE cells at least one papillary diameter away from the edge of the geographic atrophy (GA) lesion. [This invention 1045] The method of the present invention 1001, wherein the transplantation of RPE cells comprises injecting RPE cells in one or more of the following steps: covering the GA lesion, covering the fovea, covering part or all of the transition zone bordering the GA lesion, or covering surrounding healthy tissue adjacent to the GA lesion. [The present invention 1046] The method of claim 1045, wherein the transition zone comprises the area between the intact retina and the degenerated retina. [This invention 1047] The method of claim 1045, wherein the step of covering the GA lesion comprises covering the entire GA lesion with the bleb. [This invention 1048] The size of the GA is 0.1 mm 2 ~approx. 50mm 2 , about 0.5mm 2 ~about 30mm 2 , about 0.5mm 2 ~about 15mm 2 , about 0.1mm 2 ~about 10mm 2 , about 0.25mm 2 ~about 5mm 2 , or any point between the two points. [This invention 1049] 1001. The method of claim 1001, wherein administering comprises administering RPE cells such that central macular vision is preserved. [The present invention 1050] RPE cells (a) culturing human embryonic stem cells or induced pluripotent stem cells in a medium containing nicotinamide to generate differentiated cells; (b) culturing the differentiated cells in a medium containing nicotinamide and activin A to generate cells that further differentiate into the RPE lineage; and (c) culturing the cells further differentiating into the RPE lineage in a medium containing nicotinamide but not activin A. Generated by The method of the present invention 1001. [This invention 1051] The method of claim 1050, wherein the embryonic stem cells or induced pluripotent stem cells are grown in a medium comprising bFGF and TGFβ under non-adherent conditions. [This invention 1052] The method of claim 1050, wherein the medium of (a) is substantially free of activin A. [This invention 1053] 1001. The method of claim 1001, wherein said cells are administered in a single dose. [This invention 1054] 1001. The method of claim 1001, wherein said cells are administered to the subretinal space of the subject. [This invention 1055] 1002. The method of claim 1001, wherein the subretinal administration is intravitreal administration or suprachoroidal administration. [This invention 1056] 1001. The method of claim 1001, wherein the administration is by cannula. [This invention 1057] 1056. The method of claim 1056, wherein the cannula administration site heals within about 1 to about 30 days. [This invention 1058] 1056. The method of claim 1056, wherein the healing of the cannulated administration site occurs within about 5 to about 21 days or within about 7 to about 15 days. [This invention 1059] 1001. The method of claim 1001, further comprising administering immunosuppression to the subject for 1 day to 3 months after administration of the RPE cells. [The present invention 1060] The method of claim 1001, further comprising administering immunosuppression to the subject for three months after administration of the RPE cells. [This invention 1061] The method of the present invention 1001, further comprising administering immunosuppression to the subject for 1 day to 1 month after administration of the RPE cells. [This invention 1062] 1001. The method of claim 1001, wherein the retinal disease or condition is selected from the group consisting of moderate atrophic AMD, retinitis pigmentosa, retinal detachment, retinal dysplasia, retinal atrophy, retinopathy, macular dystrophy, cone dystrophy, cone-rod dystrophy, Malattia Leventinese, Doyne honeycomb dystrophy, Sorsby's dystrophy, pattern / sphenoid dystrophy, Best vitelliform dystrophy, North Carolina dystrophy, central areolar choroidal dystrophy, angioid streaks, toxic maculopathy, Stargardt's disease, pathological myopia, retinitis pigmentosa, and macular degeneration. [This invention 1063] The method of claim 1062, wherein the disease is age-related macular degeneration. [This invention 1064] The method of the present invention 1063, wherein the age-related macular degeneration is atrophic age-related macular degeneration. [This invention 1065] A method for enhancing the safety of a method for treating a subject with dry AMD, comprising administering a therapeutically effective amount of retinal pigment epithelial (RPE) cells to a subject who is not undergoing systemic immunosuppression. [The present invention 1066] The method of the present invention 1065, wherein the incidence and frequency of treatment-emergent adverse events is lower than with immunosuppression. [This invention 1067] A method for organizing the ellipsoid regions of the retina in a subject with GA, comprising administering a therapeutically effective amount of retinal pigment epithelial (RPE) cells, wherein after administration, the disorganized ellipsoid regions become organized. [The present invention 1068] 1067. The method of claim 1067, wherein recovering the ellipsoidal region comprises recovering by ellipsoidal region analysis. [The present invention 1069] The method of claim 1067, wherein the ellipsoid area analysis includes visual analysis of the ellipsoid area and comparing the subject's ellipsoid area to that of an age-matched and gender-matched control, baseline, or fellow eye. [The present invention 1070] The method of claim 1067, wherein recovery is indicated by restoration of normal structure compared to an age-matched and gender-matched control, baseline, or fellow eye. [This invention 1071] The method of the present invention 1067, wherein recovery comprises a subjective assessment that one or more of the external limiting membrane, myoid region (inner segments of photoreceptors), ellipsoid region (IS / OS junction), outer segments of photoreceptors, loss of drusen, and disappearance of reticular pseudodrusen are becoming more organized. [This invention 1072] The method of claim 1067, wherein recovery comprises a subjective assessment that one or more of the fundamental underlying layers of the retina are becoming more organized. [This invention 1073] The method of the present invention 1017, wherein the basic underlying layers of the retina that are becoming more organized include one or more of the outer limiting membrane, the myoid region (inner segments of photoreceptors), the ellipsoid region (IS / OS junction), and the outer segments of photoreceptors. [This invention 1074] The method of claim 1067, wherein the subject has a BCVA of 20 / 64 or less, 20 / 70 or less, or about 20 / 64 to about 20 / 400. [This invention 1075] The method of the present invention 1001, wherein treating or slowing the progression of retinal disease is demonstrated by recovery of visual acuity as assessed by microperimetry, and wherein recovery of visual acuity as assessed by microperimetry includes a correlation between retinal sensitivity and EZ defect in microperimetry compared to baseline. [This invention 1076] The method of the present invention 1001, wherein the recovery of vision as assessed by microperimetry comprises demonstrating that the area of ​​the retina at or near the administration site of the RPE cells comprises an improved microperimetry assessment compared to the baseline microperimetry assessment. [This invention 1077] The method of the present invention 1001, wherein treating or slowing the progression of retinal disease comprises a reduction in GA lesion growth rate by about 5% to about 20%, or about 5% to about 50%, or about 5% to about 25%, or about 5% to about 100%, or about 5% to about 10% compared to baseline or the fellow eye at one year after administration. [This invention 1078] The method of claim 1001, wherein treating or slowing the progression of retinal disease comprises one or more of: stable BCVA; no decline in low-light test performance; or no decline in sensitivity in microperimetry; or no decline in reading speed, when compared to an age-matched and gender-matched control, baseline, or fellow eye, wherein the comparison is at one or more of 1 month, 3 months, 6 months, or 1 year. [This invention 1079] A pharmaceutical composition for treating or slowing the progression of a retinal disease or disorder, comprising approximately 50,000 to 500,000 RPE cells as an active substance. [The present invention 1080] A pharmaceutical composition for stabilizing the RPE in a subject with a retinal disease or disorder, comprising approximately 50,000 to 500,000 RPE cells as an active substance. [This invention 1081] RPE cells (a) the property that at least 95% of cells co-express a pre-melanosome protein (PMEL17) and a cellular retinaldehyde-binding protein (CRALBP); and (b) the transepithelial electrical resistance of the cells is greater than 100 ohms in a subject to which the cells are administered, and the subject's retinal pigmentation is stabilized between about 90 days and about 24 months after administration; characterized by, Composition of the present invention 1080. [This invention 1082] The method of the present invention 1012, wherein the restoration of the ellipsoidal region comprises an improvement in one or more of the thickness, area, or volume measurements of the EZ-RPE. [This invention 1083] The method of the present invention 1082, wherein improvement in one or more of the thickness, area, or volume measurements of the EZ-RPE is inversely correlated with visual acuity. [This invention 1084] The method of claim 1012, wherein ellipsoidal area analysis demonstrates EZ organization by a decrease in EZ volume compared to an age-matched and gender-matched control, baseline, or fellow eye. [This invention 1085] The method of claim 1084, wherein the reduction in EZ volume comprises at least 2%, or at least 5%, or at least 7%, or at least 10%, or 1-5%, or 1-10%, or 1-50%, or 10-50%. [This invention 1086] The method of claim 1084, wherein the organization of the EZ comprises a decrease in the volume of structures in the EZ of at least 2%, at least 5%, at least 10%, about 1% to about 50% from baseline. [This invention 1087] 1001. The method of claim 1001, wherein treating or slowing the progression of a retinal disease or disorder is enhanced by cellular secretion of a trophic factor. [Brief explanation of the drawings]

[0079] The techniques described herein will be more fully understood by reference to the following drawings, which are for illustrative purposes only.

[0080] [Figure 1] FIG. 1 is an illustration of a cell-based therapy to replace and support dysfunctional and degenerating RPE in dry AMD with GA. [Figure 2A] Figure 2A is a graph of best corrected visual acuity (BCVA) measured over one year for treated eyes of Cohort 1 (Patients 1, 2, and 3 (Pt. 1, Pt. 2, Pt. 3)) treated with a dose of approximately 50,000 RPE cells. [Figure 2B] Figure 2B is a graph of best corrected visual acuity (BCVA) measured over one year in the fellow eye of Cohort 1 (Patients 1, 2, and 3 (Pt. 1, Pt. 2, Pt. 3)). [Figure 2C] Figure 2C is a graph showing BCVA over time in the treated eye. [Figure 2D] FIG. 2D is a graph showing the mean BCVA of the treated eyes. [Figure 2E] Figure 2E is a graph showing the BCVA of the fellow eye over time. [Figure 2F] Figure 2F is a graph showing the mean BCVA of the fellow eye. [Figure 3] Figure 3 shows color fundus images of Cohort 1 (Patients 1, 2, and 3 (Pt. 1, Pt. 2, Pt. 3)) at preoperative (preoperative) and intraoperative (intraoperative) time points. [Figure 4] Figure 4 shows color fundus images of Cohort 1 (Patients 1, 2, and 3 (Pt. 1, Pt. 2, Pt. 3)) before treatment with a target dose of 50,000 RPE cells (preoperative) and 2 months after treatment. [Figure 5] Figure 5 shows color fundus images of Cohort 1 (Patients 1, 2, and 3 (Pt. 1, Pt. 2, Pt. 3)) before treatment with a target dose of 50,000 RPE cells (preoperative) and 9 months to 1 year after treatment (postoperative). [Figure 6]Figure 6 shows blue autofluorescence images from patient 1 (cohort 1, treated with a dose of 50,000 RPE cells) preoperatively and at 1 day, 1 week, 2 months, 4.5 months, and 9 months postoperatively. [Figure 7] Figure 7 shows blue autofluorescence images from patient 2 preoperatively and at 1 day, 1 week, 2 months, 6 months, and 9 months postoperatively. [Figure 8] Figure 8 shows blue autofluorescence images from patient 3 preoperatively and at 1 day, 1 week, 2 months, 7 months, and 9 months postoperatively. [Figure 9] Figure 9 shows color images of patient 4 in cohort 2 (200,000 RPE cell suspension dose) at the time of surgery (day 0), FAF and color images at postoperative day 1, and color images at 2, 3, 4, and 6 months postoperatively. [Figure 10] Figure 10 shows color images and corresponding FAF images for patient 5 of cohort 2 (200,000 RPE cell suspension dose) at day 0, 1 month, 2 months, 3 months, and 6 months. [Figure 11] FIG. 11 shows OCT images of the treatment injection sites for Cohort 1. [Figure 12] Figure 12 shows OCT scans of patient 1 preoperatively and at 1 week, 1 month, and 1 year postoperatively. [Figure 13] Figure 13 shows OCT scans of patient 2 preoperatively and at 1 and 9 months postoperatively. [Figure 14] Figure 14 shows OCT scans of patient 3 preoperatively and at 3 and 9 months postoperatively. [Figure 15] Figure 15 shows OCT and infrared OCT scans of patient 4 (200,000 RPE cell suspension dose) in cohort 2 pre-operatively, and at 1 and 9 months post-operatively. [Figure 16] Figure 16 shows OCT scans of patient 5 in cohort 2 (200,000 RPE cell suspension dose) at baseline, 1 week, 2 weeks, 1 month, 2 months, 3 months and 6 months post-op. [Figure 17]FIG. 17 shows OCT scans, infrared images, and histological images following subretinal transplantation of hESC-RPE cells in pig eyes. [Figure 18] FIG. 18 shows a benign teratoma in the subretinal space of a NOD-SKID mouse. [Figure 19] FIG. 19 shows hESC-derived RPE cells in the subretinal space of NOD-SKID mice treated with 100,000 hESC-derived RPE cells in solution. [Figure 20] FIG. 20 shows HuNu+ cells in the subretinal space of NOD-SKID mice treated with 100,000 hESC-derived RPE cells in solution. [Figure 21] Figure 21 shows the engraftment and survival of hESC-derived RPE in three animal species using staining to indicate the presence of human cells. [Figure 22] Figure 22A shows a blue autofluorescence image from patient 8 (cohort 3; 100,000 RPE cells / 50 μL dose) taken before surgery, showing a baseline image of GA (dark area), the outline of the future bleb border (dotted line), and the exact implantation location (asterisk). Figure 22B shows a color fundus image from patient 8 taken before surgery, showing a baseline image of GA (dark area), the outline of the future bleb border (dotted line), and the exact implantation location (asterisk). Figure 22C shows a color image of the implanted bleb at the time of surgery. [Figure 23] FIG. 23 shows a color fundus image of patient 8 at 1 month. [Figure 24] Figure 24A shows a blue autofluorescence image taken at 1 month for patient 8. Figure 24B shows a blue autofluorescence image taken at 2 months for patient 8. Figure 24C shows a blue autofluorescence image taken at 3 months for patient 8. [Figure 25] FIG. 25 shows infrared and corresponding OCT images of the transformation zone for patient 8 at baseline (pre-surgery), 1 month, 2 months, and 3 months. [Figure 26] FIG. 26 shows infrared and corresponding OCT images of the transformation zone for patient 8 at baseline (pre-surgery), 1 month, 2 months, and 3 months. [Figure 27] FIG. 27 shows infrared and corresponding OCT images of the transformation zone for patient 8 at baseline (pre-surgery), 1 month, 2 months, and 3 months. DETAILED DESCRIPTION OF THE INVENTION

[0081] Detailed Description The RPE cell compositions and methods described herein can be used to slow the progression of retinal degenerative diseases or disorders in a subject, slow the progression of age-related macular degeneration (AMD) or intermediate AMD, prevent retinal degenerative diseases, prevent AMD, restore retinal pigment epithelium (RPE), increase RPE, replace RPE, or treat RPE diseases, defects, conditions, and / or damage by administering a composition comprising RPE cells to the subject. For example, a human embryonic stem cell-derived RPE cell composition can be injected into the subretinal space to promote RPE recovery and prevent the progression of retinal degeneration caused by a retinal disease or condition.

[0082] In certain embodiments, RPE cells are administered onto GA lesions or onto surrounding healthy tissues near GA lesions. Administration into GA lesions helps repair or correct the lesions. Administration of RPE cells into surrounding healthy tissues near GA lesions prevents further growth of the lesions.

[0083] In certain embodiments, once transplanted, RPE cell grafts secrete these factors to provide long-term trophic support to degenerating retinal tissue. This trophic support can act to attenuate retinal degeneration and vision loss in some subjects. Trophic factors are known to promote cell survival and differentiation. Examples of trophic factors and trophic factor families include, but are not limited to, neurotrophins, the ciliary neurotrophic factor / leukemia inhibitory factor (CNTF / LIF) family, the hepatocyte growth factor / scatter factor family, the insulin-like growth factor (IGF) family, and the glial cell line-derived neurotrophic factor (GDNF) family. The RPE cells described herein can begin secreting trophic factors immediately after administration or retinal transplantation. Furthermore, once the cells integrate with recipient cells and establish synaptic contacts with the target cells, a steady flow of neuroprotective support can begin.

[0084] In certain embodiments, the retinal degenerative disease can be one or more of RPE dysfunction, photoreceptor dysfunction, lipofuscin accumulation, drusen formation, or inflammation.

[0085] In other embodiments, the retinal degenerative disease is selected from at least one of retinitis pigmentosa, Leber's congenital amaurosis, hereditary or acquired macular degeneration, age-related macular degeneration (AMD), Best's disease, retinal detachment, gyrate atrophy, choroideremia, pattern dystrophy, RPE dystrophy, Stargardt's disease, and damage to the RPE and retina caused by any one of light, laser, infection, radiation, neovascularization, or traumatic injury. In yet other embodiments, the AMD is geographic atrophy (GA).

[0086] In certain embodiments, RPE defects can be caused by one or more of the following: old age, smoking, unhealthy weight, low intake of antioxidants, or cardiovascular disorders. In other embodiments, RPE defects can result from congenital abnormalities.

[0087] "Retinal pigment epithelial cells," "RPE cells," and "RPE" may be used interchangeably where the context permits, e.g., to refer to cells of a cell type that are functionally, epigenetically, or by expression profile similar to that of native RPE cells that form the pigment epithelial cell layer of the retina (e.g., when transplanted, administered, or delivered intraocularly, they exhibit similar functional activity as native RPE cells).

[0088] According to some embodiments, RPE cells express at least one, two, three, four, or five markers of mature RPE cells. According to some embodiments, RPE cells express at least two to at least ten, or at least two to at least thirty markers of mature RPE cells. Such markers include, but are not limited to, CRALBP, RPE65, PEDF, PMEL17, bestrophin 1, and tyrosinase. Optionally, RPE cells may also express a marker of RPE progenitor cells (e.g., MITF). In other embodiments, RPE cells express PAX-6. In other embodiments, RPE cells express at least one marker of retinal progenitor cells, including, but not limited to, Rx, OTX2, or SIX3. Optionally, RPE cells may express either SIX6 and / or LHX2.

[0089] As used herein, the phrase "marker of mature RPE cells" refers to an antigen (e.g., a protein) that is elevated (e.g., at least 2-fold, at least 5-fold, at least 10-fold) in mature RPE cells compared to non-RPE cells or immature RPE cells.

[0090] As used herein, the phrase "marker of RPE progenitor cells" refers to an antigen (e.g., a protein) that is elevated (e.g., at least 2-fold, at least 5-fold, at least 10-fold) in RPE progenitor cells compared to non-RPE cells.

[0091] In other embodiments, the RPE cells have a morphology similar to that of natural RPE cells that form the pigmented epithelial cell layer of the retina, e.g., the cells may be pigmented and have a characteristic polygonal shape.

[0092] According to yet another embodiment, the RPE cells can treat diseases such as macular degeneration.

[0093] According to a further aspect, the RPE cells meet at least one, two, three, four or all of the requirements listed herein above.

[0094] As used herein, the term "stem cells" refers to cells (e.g., multipotent or pluripotent stem cells) that can remain in an undifferentiated state in culture for extended periods of time until induced to differentiate into other cell types (e.g., fully differentiated cells) with specific specialized functions. Preferably, the term "stem cells" encompasses embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs), adult stem cells, mesenchymal stem cells, and hematopoietic stem cells.

[0095] According to some embodiments, the RPE cells are generated from pluripotent stem cells (eg, ESCs or iPSCs).

[0096] Induced pluripotent stem cells (iPSCs) can be generated from somatic cells by genetic manipulation, e.g., retroviral transduction of somatic cells such as fibroblasts, hepatocytes, and gastric epithelial cells with transcription factors such as Oct-3 / 4, Sox2, c-Myc, and KLF4 [Yamanaka S, Cell Stem Cell. 2007, 1(1):39-49; Aoi T, et al., Generation of Pluripotent Stem Cells from Adult Mouse Liver and Stomach Cells. Science. 2008 Feb 14. (Epub ahead of print); IH Park, Zhao R, West JA, et al. Reprogramming of human somatic cells to pluripotency with defined factors. Nature 2008; 451:141-146; K Takahashi, Tanabe K, Ohnuki M, et al. Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell 2007;131:861-872]. Other embryonic-like stem cells can be generated by nuclear transfer into oocytes, fusion with embryonic stem cells, or nuclear transfer into zygotes when the recipient cell is arrested in mitosis. Additionally, iPSCs can be generated using non-integrative methods, such as by using small molecules or RNA.

[0097] The term "embryonic stem cells" refers to embryonic cells that can differentiate into cells of all three embryonic germ layers (i.e., endoderm, ectoderm, and mesoderm) or remain undifferentiated. The term "embryonic stem cells" can include cells obtained from embryonic tissues (e.g., blastocysts) formed after conception before the implantation of an embryo (i.e., pre-implantation blastocysts), expanded blastocyst cells (EBCs) obtained from blastocysts at the post-implantation / pre-gastrulation stage (see WO2006 / 040763), and embryonic germ (EG) cells obtained from fetal reproductive tissue at any time during pregnancy, preferably before 10 weeks of pregnancy. The embryonic stem cells of some embodiments of the present disclosure can be obtained using well-known cell culture methods. For example, human embryonic stem cells can be isolated from human blastocysts.

[0098] Human blastocysts are typically obtained from human in vivo preimplantation embryos or in vitro fertilized (IVF) embryos. Alternatively, single-cell human embryos can be expanded to the blastocyst stage. For the isolation of human ES cells, the zona pellucida is removed from the blastocyst, and the inner cell mass (ICM) is isolated by a procedure in which trophectoderm cells are lysed and removed from the intact ICM by gentle pipetting. The ICM is then seeded into tissue culture flasks containing an appropriate medium that allows its proliferation. After 9–15 days, ICM-derived outgrowths are dissociated into clumps by either mechanical dissociation or enzymatic digestion, and the cells are then replated in fresh tissue culture medium. Colonies exhibiting undifferentiated morphology are individually selected with a micropipette, mechanically dissociated into clumps, and replated. The resulting ES cells are then periodically split every 4–7 days. For details on methods for preparing human ES cells, see Reubinoff et al. Nat Biotechnol 2000, May:18(5):559; Thomson et al., [U.S. Patent No. 5,843,780; Science 282:1145, 1998; Curr. Top. Dev. Biol. 38:133, 1998; Proc. Natl. Acad. Sci. USA 92:7844, 1995]; Bongso et al., [Hum Reprod 4:706, 1989]; and Gardner et al., [Fertil. Steril. 69:84, 1998].

[0099] It will be appreciated that commercially available stem cells can also be used in accordance with some aspects of the present disclosure. Human ES cells can be purchased from the NIH Human Embryonic Stem Cell Registry, www.grants.nih.govstem_cells / , or other hESC registries. Non-limiting examples of commercially available embryonic stem cell lines include HAD-C102, ESI, BGO1, BG02, BG03, BG04, CY12, CY30, CY92, CY10, TE03, TE32, CHB-4, CHB-5, CHB-6, CHB-8, CHB-9, CHB-10, CHB-11, CHB-12, HUES1, HUES2, HUES3, HUES4, HUES5, HUES6, HUES7, HUES8, HUES9, HUES10, HUES11, HUES12, HUES13, HUES14, HUES15, HUES16, HUES17, HUES18, HUES19, HUES20, HUES21, HUES22, HUES23, HUES24, HUES25, HUES26, HUES27, HUES28, HUES29, HUES30, HUES31, HUES32, HUES33, HUES34, HUES35, HUES36, HUES37, HUES38, HUES39, HUES40, HUES41, HUES42, HUES43, HUES44, HUES45, HUES46, HUES47, HUES48, HUES49, HUES50, HUES51, HUES52, HUES53, HUES54, HUES55, HUES 5, HUES16, HUES17, HUES18, HUES19, HUES20, HUES21, HUES22, HUES23, HUES24, HUES25, HUES26, HUES27, HUES28, CyT49, RUES3, WAO1, UCS F4, NYUES1, NYUES2, NYUES3, NYUES4, NYUES5, NYUES6, NYUES7, UCLA1, UCLA2, UCLA3, WA077(H7), WA09(H9), WA13(Hl3), WA14(H14), HUES 62, HUES 63, HUES 64, CT I, CT2, CT3, CT4, MA135, Eneavour-2, WIBR1, WIBR2, WIBR3, WIBR4, WIBR5, WIBR6, HUES45, Shef3, Shef6, BJNhem19, BJNhem20, SAOO1, and SAOOl.

[0100] According to some embodiments, the embryonic stem cell line is HAD-C102 or ESI.

[0101] Furthermore, ES cells have been used in mouse (Mills and Bradley, 2001), golden hamster [Doetschman et al., 1988, Dev Biol. 127:224-7], rat [Iannaccone et al., 1994, Dev Biol. 163:288-92], rabbit [Giles et al. 1993, Mol Reprod Dev. 36:130-8; Graves & Moredith, 1993, Mol Reprod Dev. 1993, 30 36:424-33], and several livestock species [Notarianni et al., 1991, J Reprod Fertil Suppl. 43:255-60; Wheeler 1994, Reprod Fertil Dev. 6:563-8; Mitalipova et al. al., 2001, Cloning. 3:59-67] and other species, including non-human primate species (rhesus monkeys and marmosets) [Thomson et al., 1995, Proc Natl Acad Sci USA. 92:7844-8; Thomson et al., 1996, Biol Reprod. 55:254-9].

[0102] Expanded blastocyst cells (EBCs) can be obtained from blastocysts at the pre-gastrulation stage, at least 9 days post-fertilization. Prior to culturing the blastocysts, the zona pellucida is digested (e.g., with Tyrode's acid solution (Sigma Aldrich, St. Louis, MO, USA)) to expose the inner cell mass. The blastocysts are then cultured in vitro as whole embryos for at least 9 and up to 14 days post-fertilization (i.e., before the gastrulation event) using standard embryonic stem cell culture methods.

[0103] Another method for preparing ES cells is described in Chung et al., Cell Stem Cell, Volume 2, Issue 2, 113-117, 7 February 2008. This method involves removing a single cell from an embryo during an in vitro fertilization process. The embryo is not destroyed in this process.

[0104] Embryonic germ cells (EG) are prepared from primordial germ cells obtained from fetuses approximately 8-11 weeks gestation (in the case of human fetuses) using laboratory techniques known to those skilled in the art. The genital ridges are dissociated, cut into small pieces, and then dissociated into cells by mechanical dissociation. The EG cells are then grown in tissue culture flasks containing the appropriate medium. The cells are cultured with daily changes of medium until a cell morphology consistent with EG cells is observed, typically after 7-30 days or 1-4 passages. For further details regarding the preparation of human EG cells, see Shamblott et al., [Proc. Natl. Acad. Sci. USA 95:13726, 1998] and U.S. Patent No. 6,090,622.

[0105] Yet another method of preparing ES cells is by parthenogenesis, again without destroying the embryo in the process.

[0106] ES cell culture methods can include the use of feeder cell layers that secrete factors required for stem cell proliferation while simultaneously inhibiting their differentiation. Culture is typically performed on solid surfaces, such as surfaces coated with gelatin or vimentin. Exemplary feeder layers include human embryonic fibroblasts, adult fallopian epithelial cells, primary mouse embryonic fibroblasts (PMEFs), mouse embryonic fibroblasts (MEFs), mouse fetal fibroblasts (MFFs), human embryonic fibroblasts (HEFs), human fibroblasts obtained from the differentiation of human embryonic stem cells, human fetal muscle cells (HFMs), human fetal skin cells (HFSs), human adult skin cells, human foreskin fibroblasts (HFFs), human umbilical cord fibroblasts, human cells obtained from the umbilical cord or placenta, and human bone marrow stromal cells (hMSCs). Growth factors may be added to the culture medium to maintain ESCs in an undifferentiated state. Such growth factors include bFGF and / or TGF. In another embodiment, agents may be added to the culture medium to maintain hESCs in a naive, undifferentiated state—see, e.g., Kalkan et al., 2014, Phil. Trans. R. Soc. B, 369:20130540.

[0107] Human umbilical cord fibroblasts can be grown in Dulbecco's modified Eagle's medium (e.g., DMEM, SH30081.01, Hyclone) supplemented with human serum (e.g., 20%) and glutamine. Preferably, the human umbilical cord cells are irradiated. This can be done using methods known in the art (e.g., Gamma cell, 220 Exel, MDS Nordion 3,500-7500 rads). After obtaining sufficient cells, they can be frozen (e.g., cryopreserved). For ESC expansion, human umbilical fibroblasts are typically seeded at a concentration of approximately 25,000–100,000 cells / cm in DMEM (e.g., SH30081.01, Hyclone) supplemented with approximately 20% human serum (and glutamine) onto a solid surface (e.g., T75 or T175 flasks) optionally coated with an adhesive substrate such as gelatin (e.g., recombinant human gelatin (RhG 100-001, Fibrogen) or human vitronectin or laminin 521 (Bio lamina)). hESCs are typically plated onto feeder cells after 1–4 days in support medium (e.g., NUTRISTEM® or NUT(+) containing human serum albumin). Additional factors may be added to the medium to prevent ESC differentiation, such as bFGF and TGFβ. After obtaining a sufficient amount of hESCs, the cells can be mechanically disrupted (e.g., using a sterile tip or a disposable sterile stem cell tool; 14602 Alternatively, cells may be removed by enzymatic treatment (e.g., collagenase A, or TrypLE Select). This process may be repeated several times to reach the required amount of hESCs. According to some embodiments, after the first round of expansion, hESCs are removed using TrypLE Select, and after the second round of expansion, hESCs are removed using collagenase A.

[0108] ESCs can be grown on feeders before the differentiation stage. Exemplary feeder layer-based cultures are described herein above. Growth is typically carried out for at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 days. Growth is carried out for at least 1 passage, at least 2 passages, at least 3 passages, at least 4 passages, at least 5 passages, at least 6 passages, at least 7 passages, at least 8 passages, at least 9 passages, or at least 10 passages. In some embodiments, growth is carried out for at least 2 passages to at least 20 passages. In other embodiments, growth is carried out for at least 2 passages to at least 40 passages. After growth, pluripotent stem cells (e.g., ESCs) are subjected to differentiation induction using a differentiation agent.

[0109] Feeder-free systems have also been used in ES cell culture, utilizing matrices supplemented with serum replacement, cytokines, and growth factors (including IL6 and soluble IL6 receptor chimeras) as an alternative to feeder cell layers. Stem cells can be grown on solid surfaces, such as extracellular matrices (e.g., MATRIGELR™, laminin, or vitronectin), in the presence of media such as Lonza L7 System, mTeSR, StemPro, XFKSR, E8, or NUTRISTEM®. Unlike feeder-based cultures, which require simultaneous growth of feeder cells and stem cells and can result in mixed cell populations, stem cells grown in feeder-free systems are easily detached from the surface. The media used for stem cell growth contain factors that effectively inhibit differentiation and promote proliferation, such as MEF-conditioned medium and bFGF.

[0110] In some embodiments, after expansion, the pluripotent ESCs are subjected to differentiation induction on an adhesive surface (without the intermediate formation of spheroids or enviroid bodies). See, e.g., WO2017 / 072763, which is incorporated herein by reference in its entirety.

[0111] Thus, according to one aspect of the present disclosure, at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the cells subjected to differentiation induction on an adhesive surface are undifferentiated ESCs and express markers of pluripotency. For example, at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the cells are Oct4+. + TRA-1-60 + Undifferentiated ESCs may express other markers of pluripotency, such as NANOG, Rex-1, alkaline phosphatase, Sox2, TDGF-β, SSEA-3, SSEA-4, and / or TRA-1-81.

[0112] In one exemplary differentiation protocol, undifferentiated embryonic stem cells are differentiated to the RPE cell lineage on an adhesive surface using a first differentiation agent, and then further differentiated into RPE cells using a member of the transforming growth factor B (TGFB) superfamily (e.g., TGF1, TGF2, and TGF3 subtypes, as well as cognate ligands including activins (e.g., activin A, activin B, and activin AB), nodal, anti-Müllerian hormone (AMH), certain bone morphogenetic proteins (BMPs), such as BMP2, BMP3, BMP4, BMP5, BMP6, and BMP7, and growth differentiation factors (GDFs). According to a specific embodiment, the member of the transforming growth factor B (TGFB) superfamily is activin A, e.g., 20-200 ng / ml, e.g., 100-180 ng / ml.

[0113] According to some embodiments, the first differentiation agent is nicotinamide (NA) used at a concentration of about 1-100 mM, 5-50 mM, 5-20 mM, e.g., 10 mM. According to other embodiments, the first differentiation agent is 3-aminobenzmine.

[0114] NA, also known as "niacinamide," is an amide derivative of vitamin B3 (niacin) that is believed to maintain and improve beta cell function. NA has the chemical formula C6H6N20. NA is essential for growth and the conversion of food to energy and is used in the treatment of arthritis and the treatment and prevention of diabetes. TIFF2025161869000001.tif29128

[0115] According to some embodiments, the nicotinamide is a nicotinamide derivative or a nicotinamide mimetic. As used herein, the term "nicotinamide (NA) derivative" refers to a compound that is a chemically modified derivative of natural NA. In one embodiment, the chemical modification may be a substitution of the pyridine ring of the basic NA structure (via a carbon or nitrogen member of the ring) via the nitrogen or oxygen atom of the amide moiety. When substituted, one or more hydrogen atoms may be replaced by a substituent, and / or the substituent may be bonded to the N atom to form a tetravalent positively charged nitrogen. Thus, the nicotinamide of the present invention includes substituted or unsubstituted nicotinamide. In another embodiment, the chemical modification may be the deletion or substitution of a single group, for example, to form a thiobenzamide analog of NA, all of which are understood by those skilled in organic chemistry. Derivatives in the context of the present invention also include nucleoside derivatives of NA (e.g., nicotinamide adenine). Various derivatives of NA have been described, some of which are related to the inhibitory activity of PDE4 enzyme (WO03 / 068233; WO02 / 060875; GB2327675A) or as VEGF receptor tyrosine kinase inhibitor (WO1 / 55114).For example, the method of preparing 4-aryl-nicotinamide derivatives (WO05 / 014549).Other exemplary nicotinamide derivatives are disclosed in WO1 / 55114 and EP2128244.

[0116] Nicotinamide mimetics include modified forms of nicotinamide and chemical analogs of nicotinamide that replicate the action of nicotinamide in the differentiation and maturation of RPE cells from pluripotent cells.Exemplary nicotinamide mimetics include benzoic acid, 3-aminobenzoic acid, and 6-aminonicotinamide.Another class of compounds that can act as nicotinamide mimetics is the inhibitor of poly(ADP-ribose) polymerase (PARP).Exemplary PARP inhibitors include 3-aminobenzamide, iniparib (BSI201), olaparib (AZD-2281), rucaparib (AG014699, PF-01367338), veliparib (ABT-888), CEP9722, MK4827, and BMN-673.

[0117] Additional contemplated differentiation agents include, for example, noggin, Wnt antagonists (Dkk1 or IWR1e), nodal antagonists (Lefty-A), retinoic acid, taurine, GSK3b inhibitors (CHIR99021), and notch inhibitors (DAPT).

[0118] According to a particular embodiment, differentiation is carried out as follows: (a) culturing ESCs in a medium containing a first differentiation agent (e.g., nicotinamide); and (b) culturing the cells obtained in step a) in a medium containing a member of the TGFB superfamily (e.g., activin A) and the first differentiation agent (e.g., nicotinamide).

[0119] Step (a) may be carried out in the absence of a member of the TGFβ superfamily (eg, activin A).

[0120] In some embodiments, the medium of step (a) is completely free of members of the TGFβ superfamily, hi other embodiments, the level of TGFβ superfamily members in the medium is less than 20 ng / ml, less than 10 ng / ml, less than 1 ng / ml, or even less than 0.1 ng / ml.

[0121] The above protocol may be continued by culturing the cells obtained in step (b) in a medium containing a first differentiation agent (e.g., nicotinamide) but not a member of the TGFβ superfamily (e.g., activin A). This step is referred to herein as step (b). * ) is called.

[0122] The above protocol will now be described in more detail with additional aspects. Step (a): After obtaining a sufficient amount of ESCs, the differentiation process begins. Cells can be removed from the cell culture (e.g., by using collagenase A, dispase, TrypLE select, or EDTA) and plated onto a non-adhesive substrate (e.g., a cell culture plate such as a Hydrocell or agarose-coated culture dish, or a Petri dish) in the presence of nicotinamide (and the absence of activin A). Exemplary concentrations of nicotinamide are 0.01-100 mM, 0.1-100 mM, 0.1-50 mM, 5-50 mM, 5-20 mM, and 10 mM. Once the cells are plated onto a non-adhesive substrate (e.g., a cell culture plate), the cell culture can be referred to as a cell suspension, preferably free-floating clusters in suspension culture, i.e., aggregates of cells derived from human embryonic stem cells (hESCs). The cell clusters do not adhere to any substrate (e.g., a culture plate, a carrier). Sources of free-floating stem cells have been previously described in WO 06 / 070370, which is incorporated herein by reference in its entirety. This step can be carried out for at least one day, more preferably two, three, one week, or even 14 days. Preferably, the cells are cultured in suspension for no more than three weeks, e.g., with nicotinamide at 0.01-100 mM, 0.1-100 mM, 0.1-50 mM, 5-50 mM, 5-20 mM, e.g., 10 mM (and in the absence of activin A). In one embodiment, the cells are cultured in suspension for 6-8 days, e.g., with nicotinamide at 0.01-100 mM, 0.1-100 mM, 0.1-50 mM, 5-50 mM, 5-20 mM, e.g., 10 mM (and in the absence of activin A).

[0123] According to some embodiments, when cells are cultured on a non-adherent substrate, e.g., a cell culture plate, atmospheric oxygen conditions are 20%. However, it is also contemplated to manipulate atmospheric oxygen conditions so that the percentage of atmospheric oxygen is less than about 20%, 15%, 10%, 9%, 8%, 7%, 6%, or even less than about 5% (e.g., 1%-20%, 1%-10%, or 0-5%). According to other embodiments, cells are cultured on a non-adherent substrate initially under normal atmospheric oxygen conditions, then reduced to less than normal atmospheric oxygen conditions.

[0124] Examples of non-adherent cell culture plates include those manufactured by Nunc (eg, Hydrocell catalog number 174912).

[0125] Typically, a cluster contains at least 50-500,000, 50-100,000, 50-50,000, 50-10,000, 50-5,000, or 50-1,000 cells. According to one embodiment, the cells within the cluster are not organized into layers but form irregular shapes. In one embodiment, the cluster is substantially free of pluripotent embryonic stem cells. In another embodiment, the cluster contains a small number of pluripotent embryonic stem cells (e.g., 5% or less or 3% or less (e.g., 0.01-2.7%) of cells co-expressing OCT4 and TRA-1-60 at the protein level). Typically, the cluster contains cells that have partially differentiated under the influence of nicotinamide. Such cells primarily express neural and retinal progenitor markers, such as PAX6, Rax, Six3, and / or CHX10.

[0126] Clusters can be dissociated using enzymatic or non-enzymatic methods known in the art (e.g., mechanical). According to some embodiments, the cells are dissociated so that they are no longer clusters, e.g., aggregates or clumps of 2-100,000 cells, 2-50,000 cells, 2-10,000 cells, 2-5,000 cells, 2-1,000 cells, 2-500 cells, 2-100 cells, or 2-50 cells. According to certain embodiments, the cells are in a single cell suspension.

[0127] The cells (e.g., dissociated cells) are then seeded onto an adhesive substrate and cultured in the presence of, for example, 0.01 to 100 mM, 0.1 to 100 mM, 0.1 to 50 mM, 5 to 50 mM, 5 to 20 mM, or 10 mM nicotinamide (and in the absence of activin A). This step can be carried out for at least 1 day, more preferably 2 days, 3 days, 1 week, or even 14 days. Preferably, the cells are cultured in the presence of nicotinamide (and in the absence of activin A) for no more than 3 weeks. In an exemplary embodiment, this step is carried out for 6 to 7 days.

[0128] In other embodiments, when cells are cultured on an adhesive substrate, such as laminin, the atmospheric oxygen conditions are 20%. The atmospheric oxygen conditions may be manipulated to provide a percentage of atmospheric oxygen of less than about 20%, 15%, or 10%, more preferably less than about 9%, less than about 8%, less than about 7%, or less than about 6%, and more preferably about 5% (e.g., 1%-20%, 1%-10%, or 0-5%).

[0129] According to some embodiments, the cells are first cultured on the adherent substrate under normal atmospheric oxygen conditions, and then the oxygen is reduced to below normal atmospheric oxygen conditions.

[0130] Examples of adhesive substrates or mixtures of substances include, but are not limited to, fibronectin, laminin, poly-D-lysine, collagen, and gelatin.

[0131] Step (b): After the first stage of differentiation induction (step a; i.e., culturing in the presence of nicotinamide (e.g., 0.01 to 100 mM, 0.1 to 100 mM, 0.1 to 50 mM, 5 to 50 mM, 5 to 20 mM, e.g., 10 mM)), the partially differentiated cells can then be subjected to a further differentiation stage on an adhesive substrate by culturing them in the presence of activin A (e.g., 0.01 to 1000 ng / ml, 0.1 to 200 ng / ml, 1 to 200 ng / ml, e.g., 140 ng / ml, 150 ng / ml, 160 ng / ml, or 180 ng / ml). Activin A can therefore be added at a final molar concentration of 0.1 pM to 10 nM, 10 pM to 10 nM, 0.1 nM to 10 nM, 1 nM to 10 nM, e.g., 5.4 nM.

[0132] Nicotinamide may also be added at this stage (e.g., 0.01 to 100 mM, 0.1 to 100 mM, 0.1 to 50 mM, 5 to 50 mM, 5 to 20 mM, e.g., 10 mM). This stage may be carried out for 1 day to 10 weeks, 3 days to 10 weeks, 1 week to 10 weeks, 1 week to 8 weeks, or 1 week to 4 weeks, e.g., at least 1 day, at least 2 days, at least 3 days, at least 5 days, at least 1 week, at least 9 days, at least 10 days, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks, at least 7 weeks, at least 8 weeks, at least 9 weeks, or at least 10 weeks.

[0133] According to some embodiments, this step is carried out for about 8 days to about 2 weeks. As detailed hereinabove, this differentiation step may be carried out under low or normal atmospheric oxygen conditions.

[0134] Process (b *After step (b), the differentiated cells are optionally subjected to a subsequent differentiation step on an adhesive substrate, i.e., culturing in the presence of nicotinamide and activin A on the adhesive substrate, in the presence of nicotinamide (e.g., 0.01-100 mM, 0.1-100 mM, 0.1-50 mM, 5-50 mM, 5-20 mM, e.g., 10 mM), but in the absence of activin A. This step may be carried out for at least 1 day, 2 days, 5 days, at least 1 week, at least 2 weeks, at least 3 weeks, or even 4 weeks. As detailed herein above, this differentiation step may also be carried out in low or normal atmospheric oxygen conditions.

[0135] The basal medium in which ESCs differentiate can be any known cell culture medium known in the art for supporting cell growth in vitro, typically a medium containing a defined base solution containing salts, sugars, amino acids, and any other nutrients necessary to maintain cells in a viable state during culture. According to certain embodiments, the basal medium is not a conditioned medium. Non-limiting examples of commercially available basal media that can be utilized in accordance with the present invention include NUTRISTEM® (without bFGF and TGF for ESC differentiation, and with bFGF and TGF for ESC proliferation), NEUROBASAL™, KO-DMEM, DMEM, DMEM / F12, CELLGRO™ Stem Cell Growth Medium, or X-VIVO™. The basal medium may be supplemented with various agents known in the art for cell culture. The following are non-limiting references to various supplements that may be included in cultures used in accordance with the present disclosure: serum or serum replacements, such as, but not limited to, Knockout Serum Replacement (KOSR), NUTRIDOMA-CS, TCH™, N2, N2 derivatives, or B27, or combinations; extracellular matrix (ECM) components, such as, but not limited to, fibronectin, laminin, collagen, and gelatin. The ECM may be used to deliver one or more members of the TGF-β superfamily of growth factors; antimicrobial agents, such as, but not limited to, penicillin and streptomycin; and non-essential amino acids (NEAAs), neurotrophins known to play a role in promoting survival of SCs in culture, such as, but not limited to, BDNF, NT3, and NT4.

[0136] According to some embodiments, the medium used to differentiate the ESCs is NUTRISTEM® medium (Biological Industries, 06-5102-01-1A).

[0137] According to some embodiments, the differentiation and proliferation of ESCs is carried out under xeno-free conditions. According to other embodiments, the proliferation / growth medium is substantially free of xenocontaminants, i.e., free of animal-derived components such as serum, animal-derived growth factors, and albumin. Thus, according to these embodiments, the culture is carried out in the absence of xenocontaminants. Other methods for culturing ESCs under xeno-free conditions are provided in U.S. Patent Application No. 20130196369, the entire contents of which are incorporated herein by reference.

[0138] Preparations comprising RPE cells may be prepared in accordance with Good Manufacturing Practices (GMP) (e.g., the preparation is GMP compliant) and / or Current Good Tissue Practices (GTP) (e.g., the preparation may be GTP compliant).

[0139] During the differentiation step, the differentiation state of the embryonic stem cells can be monitored. Cell differentiation can be determined by examination of cell or tissue specific markers known to be indicative of differentiation.

[0140] Tissue / cell-specific markers can be detected using immunological techniques well known in the art [Thomson JA et al., (1998). Science 282:1145-7]. Examples include, but are not limited to, flow cytometry for membrane-bound or intracellular markers, immunohistochemistry for extracellular and intracellular markers, and enzyme-linked immunosorbent assay for secreted molecular markers.

[0141] After the differentiation steps described herein above, a mixed cell population can be obtained that contains both pigmented and non-pigmented cells. According to this aspect, the cells of the mixed cell population are removed from the plate. In some embodiments, this is done enzymatically (e.g., using trypsin (TrypLE Select); see, e.g., WO2017 / 021973, incorporated herein by reference in its entirety). According to this aspect of the invention, at least 10%, 20%, 30%, at least 40%, at least 50%, at least 60%, or at least 70% of the cells removed from the culture (and subsequently expanded) are non-pigmented cells. In other embodiments, this is done mechanically, e.g., using a cell scraper. In yet other embodiments, this is done chemically (e.g., with EDTA). Combinations of enzymatic and chemical treatments are also contemplated. For example, EDTA and enzymatic treatments can be used. Furthermore, at least 10%, 20%, or even 30% of the cells removed from the culture (and subsequently expanded) can be pigmented cells.

[0142] According to one aspect of the disclosure, at least 50%, 60%, 70%, 80%, 90%, 95%, 100% of all cells in the culture are removed and then expanded.

[0143] Mixed populations of cells can be grown on extracellular matrices such as gelatin, collagen I, collagen IV, laminin (e.g., laminin 521), fibronectin, and poly-D-lysine. For growth, cells can be cultured in serum-free KOM, serum-containing medium (e.g., DMEM containing 20% ​​human serum), or NUTRISTEM® medium (06-5102-01-1A, Biological Industries). Under these culture conditions, after passage under appropriate conditions, the ratio of pigmented cells to non-pigmented cells increases, resulting in a purified population of RPE cells. Such cells exhibit the characteristic polygonal morphology and pigmentation of RPE cells.

[0144] In one aspect, growth is carried out in the presence of nicotinamide (e.g., 0.01-100 mM, 0.1-100 mM, 0.1-50 mM, 5-50 mM, 5-20 mM, e.g., 10 mM) and in the absence of activin A.

[0145] Mixed populations of cells can be grown in suspension (with or without microcarriers) or in monolayers. Growth of mixed populations of cells in monolayer or suspension cultures can be adapted for large-scale growth in bioreactors or multi / hyperstacks by methods well known to those skilled in the art.

[0146] According to some embodiments, the expansion step is carried out for at least 1 to 20 weeks, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks, at least 7 weeks, at least 8 weeks, at least 9 weeks, or even 10 weeks. Preferably, the expansion step is carried out for 1 to 10 weeks, more preferably 2 to 10 weeks, more preferably 3 to 10 weeks, more preferably 4 to 10 weeks, or 4 to 8 weeks.

[0147] According to yet other embodiments, the mixed cell population is passaged at least once during the expansion stage, at least twice during the expansion stage, at least three times during the expansion stage, at least four times during the expansion stage, at least five times during the expansion stage, or at least six times during the expansion stage.

[0148] The inventors have shown that when cells are enzymatically harvested, it is possible to continue proliferation for more than 8, 9, or even 10 passages (e.g., 11-15 passages). The number of total cell doublings can be increased to more than 30, e.g., 31, 32, 33, 34, or more (see WO2017 / 021973, incorporated herein by reference in its entirety).

[0149] Populations of RPE cells generated according to the methods described herein can be characterized according to several different parameters. Thus, for example, the resulting RPE cells can be polygonal in shape and pigmented.

[0150] It should be understood that the cell population and cell composition disclosed herein generally lack undifferentiated human embryonic stem cells.According to some embodiments, for example, when measured by FACS, less than 1:250,000 cells are Oct4+TRA-1-60+ cells.Cells can also have downregulated (more than 5,000 times) the expression of GDF3 or TDGF when measured by PCR.In this aspect, RPE cells do not substantially express embryonic stem cell markers.The one or more embryonic stem cell markers can include OCT-4, NANOG, Rex-1, alkaline phosphatase, Sox2, TDGF-β, SSEA-3, SSEA-4, TRA-1-60, and / or TRA-1-81.

[0151] Therapeutic RPE cell preparations can be substantially purified compared to non-RPE cells and can contain at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% RPE cells. RPE cell preparations can be essentially free of non-RPE cells or consist of RPE cells. For example, a substantially purified preparation of RPE cells can contain less than about 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% of non-RPE cell types. For example, RPE cell preparations have been shown to be approximately 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.09%, 0.08%, 0.07%, 0.06%, 0.05%, 0.04%, 0.03%, 0.02%, 0.0 It may contain less than 1%, 0.009%, 0.008%, 0.007%, 0.006%, 0.005%, 0.004%, 0.003%, 0.002%, 0.001%, 0.0009%, 0.0008%, 0.0007%, 0.0006%, 0.0005%, 0.0004%, 0.0003%, 0.0002%, or 0.0001% non-RPE cells.

[0152] RPE cell preparation can be substantially pure compared to both non-RPE cells and RPE cells of other levels of maturity.The preparation can be substantially purified compared to non-RPE cells, and can be enriched for mature RPE cells.For example, in the RPE cell preparation enriched for mature RPE cells, at least about 30%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99% or 100% of RPE cells are mature RPE cells.The preparation can be substantially purified compared to non-RPE cells, and can be enriched for differentiated RPE cells, not mature RPE cells. For example, at least about 30%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the RPE cells can be differentiated RPE cells rather than mature RPE cells.

[0153] The preparations described herein are believed to be substantially free of bacterial, viral, or fungal contamination or infection, including, but not limited to, the presence of HIV1, HIV2, HBV, HCV, HAV, CMV, HTLV1, HTLV2, parvovirus B19, Epstein-Barr virus, or herpesvirus types 1 and 2, SV40, HHV5, 6, 7, 8, CMV, polyomavirus, HPV, enterovirus. The preparations described herein are believed to be substantially free of mycoplasma contamination or infection.

[0154] Another method for characterizing the cell populations disclosed herein is by marker expression. Thus, for example, at least 80%, 85%, 90%, 95%, or 100% of the cells may express bestrophin 1, as measured by immunostaining. According to one embodiment, 80-100% of the cells express bestrophin 1.

[0155] According to other embodiments, at least 80%, 85%, 87%, 89%, 90%, 95%, 97%, or 100% of the cells express microphthalmia-associated transcription factor (MITF) as measured by immunostaining, e.g., 80-100% of the cells express MITF.

[0156] According to other embodiments, as measured by immunostaining, at least 80%, 85%, 87%, 89%, 90%, 95%, 97%, or 100% of the cells express both microphthalmia-associated transcription factor (MITF) and bestrophin 1. For example, 80-100% of the cells co-express MITF and bestrophin 1.

[0157] According to other embodiments, at least 80%, 85%, 87%, 89%, 90%, 95%, 97%, or 100% of the cells express both microphthalmia-associated transcription factor (MITF) and Z0-1 as measured by immunostaining, e.g., 80-100% of the cells co-express MITF and Z0-1.

[0158] According to other embodiments, as measured by immunostaining, at least 80%, 85%, 87%, 89%, 90%, 95%, 97%, or 100% of the cells express both Z0-1 and bestrophin 1. For example, 80-100% of the cells co-express Z0-1 and bestrophin 1.

[0159] According to another embodiment, at least 50%, 60%, 70%, 80%, 85%, 87%, 89%, 90%, 95%, 97%, or 100% of the cells express paired box gene 6 (PAX-6) as measured by immunostaining or FACS, e.g., at least 50% to 100% of the cells express paired box gene 6 (PAX-6).

[0160] In another embodiment, at least 80%, 85%, 87%, 89%, 90%, 95%, 97%, or 100% of the cells express cellular retinaldehyde-binding protein (CRALBP) as measured by immunostaining, e.g., 80-100% of the cells express CRALBP.

[0161] According to another embodiment, at least 80%, 85%, 87%, 89%, 90%, 95%, 97%, or 100% of the cells express the cellular melanocyte lineage-specific antigen GP100 (PMEL17) as measured by immunostaining, e.g., about 80-100% of the cells express PMEL17.

[0162] RPE cells may co-express markers indicative of terminal differentiation, such as bestrophin 1, CRALBP, and / or RPE65. According to one embodiment, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 100%, or even about 50%-100% of the cells in the resulting RPE cell population co-express both pre-melanosome protein (PMEL17) and cellular retinaldehyde-binding protein (CRALBP).

[0163] According to a particular embodiment, the cells co-express PMEL17 (SwissProt No. P40967) and at least one polypeptide selected from the group consisting of cellular retinaldehyde-binding protein (CRALBP; SwissProt No. P12271), lecithin retinol acyltransferase (LRAT; SwissProt No. 095327), and sex-determining region Y-box 9 (SOX 9; P48436).

[0164] According to a particular aspect, at least 80% of the cells of the population express detectable levels of PMEL17 and one of the above polypeptides (e.g., CRALBP), more preferably at least 85% of the cells of the population express detectable levels of PMEL17 and one of the above polypeptides (e.g., CRALBP), more preferably at least 90% of the cells of the population express detectable levels of PMEL17 and one of the above polypeptides (e.g., CRALBP), more preferably at least 95% of the cells of the population express detectable levels of PMEL17 and one of the above polypeptides (e.g., CRALBP), and more preferably 100% of the cells of the population express detectable levels of PMEL17 and one of the above polypeptides (e.g., CRALBP as assayed by a method known to those skilled in the art (e.g., FACS)).

[0165] According to another aspect, the level of co-expression of CRALBP and one of the above polypeptides (e.g., PMEL17) (e.g., as measured by mean fluorescence intensity) is increased by at least 2-fold, more preferably at least 3-fold, more preferably at least 4-fold, even more preferably at least 5-fold, at least 10-fold, at least 20-fold, at least 30-fold, at least 40-fold, or at least 50-fold compared to undifferentiated ESCs.

[0166] In one embodiment, the RPE are terminally differentiated and generally do not express Pax6, hi another embodiment, the RPE cells are terminally differentiated and generally express Pax6.

[0167] The RPE cells described herein can also function as functional RPE cells after transplantation, forming a monolayer between the neurosensory retina and choroid of patients receiving the transplanted cells. RPE cells can also provide nutrients to neighboring photoreceptors and dispose of shed photoreceptor outer segments by phagocytosis.

[0168] According to one embodiment, the transepithelial electrical resistance of the monolayer of cells is greater than 100 ohms.

[0169] Preferably, the transepithelial electrical resistance of the cells is greater than 150, 200, 250, 300, 300, 400, 500, 600, 700, 800 ohms, or even greater than 900 ohms.

[0170] Devices for measuring transepithelial electrical resistance (TEER) are known in the art and include, for example, the EVOM2 epithelial voltmeter (World Precision Instruments).

[0171] After the expansion step, a cell population containing RPE cells is obtained, of which at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or even 100% are CRALBP+PMEL17+.

[0172] Those skilled in the art will appreciate that RPE cell induction is highly beneficial. RPE cells can be used as an in vitro model for the development of new drugs that promote their survival, regeneration, and function. RPE cells can be useful for high-throughput screening of compounds that have toxic or regenerative effects on RPE cells. They can be used to identify mechanisms, new genes, soluble, or membrane-bound factors important for the development, differentiation, maintenance, survival, and function of photoreceptor cells.

[0173] The RPE cells described herein may also serve as an unlimited source of RPE cells for transplantation, replacement, and support of dysfunctional or degenerating RPE cells in retinal degeneration and other degenerative disorders. Furthermore, genetically modified RPE cells may serve as vectors for the delivery and expression of genes in the eye and retina after transplantation.

[0174] In certain embodiments, RPE cell compositions may be produced according to the following method: (1) culturing hESCs on hUCF in CW plates in NUT+ with human serum albumin (HSA) for 2 weeks; (2) mechanically passaging hESCs to expand on hUCF in CW plates in NUT+ with HSA for 4-5 weeks (or until desired quantity of cells); (3) continuing to grow hESC colonies (e.g., using collagenase) on hUCF in 6 cm plates in NUT+ with HSA for an additional week; (4) culturing hESCs on hUCF in 6 cm plates in NUT+ with HSA for an additional week; (5) Prepare spheroid bodies (SB) by transferring colonies from approximately five 6 cm plates onto one HydroCell plate in NUT- containing nicotinamide (NIC) for approximately one week; (6) Flatten SB on Lam511 by transferring SB to two to three wells of a 6-well plate in NUT- containing NIC for approximately one week; (7) Cultivate adherent cells on Lam511 in NUT- containing NIC and activin for approximately one to two weeks, then replace the medium with NUT- containing NIC for one to three weeks; (8) Enrich pigment cells using enzymes such as TrypLE Select; (9) Grow RPE cells on gelatin in flasks in NUT- with 20% human serum for approximately two to nine weeks (with medium changes); and (10) Harvest RPE cells.

[0175] Harvesting of the expanded population of RPE cells can be performed using methods known in the art (e.g., using enzymes such as trypsin, or chemically using EDTA, etc.). In some embodiments, the RPE cells can be washed using an appropriate solution such as PBS or BSS plus. In other embodiments, the RPE cells can be filtered before being formulated into an RPE cell composition for cryopreservation and administration to a subject immediately after thawing.

[0176] After harvesting, the expanded population of RPE cells can be formulated into a specific therapeutic dose (e.g., cell number) and cryopreserved for shipment to the clinic. The ready-to-administer (RTA) RPE cell therapy composition can then be administered directly without further processing after thawing. Examples of media suitable for cryopreservation include, but are not limited to, 90% human serum / 10% DMSO, Medium 3 10% (CS10), Medium 2 5% (CS5), and Medium 1 2% (CS2), Stem Cell Banker, PRIME XV® FREEZIS, HYPOTHERMASOL®, trehalose, etc.

[0177] RPE cells formulated in a cryopreservation medium suitable for ready-to-thaw (RTA) use can include adenosine, dextran □40, lactobionic acid, HEPES (N□(2□hydroxyethyl)piperazine□N'□(2□ethanesulfonic acid)), sodium hydroxide, L-glutathione, potassium chloride, potassium bicarbonate, potassium phosphate, dextrose, sucrose, mannitol, calcium chloride, magnesium chloride, potassium hydroxide, sodium hydroxide, dimethyl sulfoxide (DMSO), and RPE cells suspended in water. An example of this cryopreservation medium is commercially available under the trade name CRYOSTOR® and manufactured by BioLife Solutions, Inc.

[0178] In a further embodiment, the cryopreservation medium includes a purine nucleoside (e.g., adenosine), a branched glucan (e.g., dextran □40), a zwitterionic organic chemical buffer (e.g., HEPES (N□(2□hydroxyethyl)piperazine□N'□(2□ethanesulfonic acid))), and a polar aprotic solvent tolerated by the cells (e.g., dimethyl sulfoxide (DMSO)). In yet a further embodiment, one or more of the purine nucleoside, branched glucan, buffer, and polar aprotic solvent are generally recognized as safe by the U.S. FDA.

[0179] In some embodiments, the cryopreservation medium further comprises one or more of a sugar acid (e.g., lactobionic acid), one or more bases (e.g., sodium hydroxide, potassium hydroxide), an antioxidant (e.g., L-glutathione), one or more halide salts (e.g., potassium chloride, sodium chloride, magnesium chloride), a basic salt (e.g., potassium bicarbonate), a phosphate salt (e.g., potassium phosphate, sodium phosphate, potassium phosphate), one or more sugars (e.g., dextrose, sucrose), a sugar alcohol (e.g., mannitol), and water.

[0180] In other embodiments, one or more of the sugar acids, bases, halide salts, basic salts, antioxidants, phosphate salts, sugars, sugar alcohols are generally recognized as safe by the US FDA.

[0181] DMSO can be used as a cryoprotectant to prevent the formation of ice crystals that can kill cells during the cryopreservation process. In some embodiments, the cryopreservable RPE cell therapy composition contains about 0.1% to about 2% DMSO (v / v). In some embodiments, the RTA RPE cell therapy composition contains about 1% to about 20% DMSO. In some embodiments, the RTA RPE cell therapy composition contains about 2% DMSO. In some embodiments, the RTA RPE cell therapy composition contains about 5% DMSO.

[0182] In some embodiments, RPE cell therapy formulated in a cryopreservation medium suitable for immediate administration after thawing may include RPE cells suspended in a DMSO-free cryopreservation medium. For example, an RTA RPE cell therapy composition may include RPE cells suspended in Trolox, Na+, K+, Ca2+, Mg2+, Cl-, H2P04-, HEPES, lactobionate, sucrose, mannitol, glucose, dextran-40, adenosine, glutathione without DMSO (dimethyl sulfoxide, (CH3)2SO), or any other dipolar aprotic solvent. An example of this cryopreservation medium is commercially available under the trade name HYPOTHERMOSOL® or HYPOTHERMOSOL®-FRS and manufactured by BioLife Solutions, Inc. In other embodiments, RPE cell compositions formulated in a cryopreservation medium suitable for immediate administration after thawing may include RPE cells suspended in trehalose.

[0183] The RTA RPE cell therapy composition may optionally include additional factors that support RPE engraftment, integration, survival, efficacy, etc. In some embodiments, the RTA RPE cell therapy composition includes an activator of the function of the RPE cell preparation described herein. In some embodiments, the RTA RPE cell therapy composition includes nicotinamide. In some embodiments, the RTA RPE cell therapy composition includes nicotinamide at a concentration of about 0.01-100 mM, 0.1-100 mM, 0.1-50 mM, 5-50 mM, 5-20 mM, e.g., 10 mM. In other embodiments, the RTA RPE cell therapy composition includes retinoic acid. In some embodiments, the RTA RPE cell therapy composition includes retinoic acid at a concentration of about 0.01-100 mM, 0.1-100 mM, 0.1-50 mM, 5-50 mM, 5-20 mM, e.g., 10 mM.

[0184] In some embodiments, RTA RPE cell therapy compositions can be formulated to include activators of various integrins that have been shown to increase attachment of RPE cell preparations, such as those described herein, to branch membranes. For example, in some embodiments, the RTA RPE cell therapy composition includes extracellular manganese (Mn2+) at a concentration of about 5 μM to 1,000 μM. In other embodiments, the RTA RPE cell therapy composition includes the conformation-specific monoclonal antibody, TS2 / 16.

[0185] In other embodiments, the RTA RPE cell therapy composition may also be formulated to include an activator of the immunomodulatory activity of RPE cells.

[0186] In some embodiments, the RTA RPE cell therapy composition may include a ROCK inhibitor.

[0187] In some embodiments, RPE cell therapy agents formulated in cryopreservation media suitable for immediate administration after thawing may include one or more immunosuppressant compounds. In certain embodiments, RPE cell therapy agents formulated in cryopreservation media suitable for immediate administration after thawing may include one or more immunosuppressant compounds formulated for sustained release of one or more immunosuppressant compounds. Immunosuppressant compounds for use with the formulations described herein may belong to the following classes of immunosuppressants: glucocorticoids, cytostatics (e.g., alkylating agents or antimetabolites), antibodies (polyclonal or monoclonal), drugs acting on immunophilins (e.g., cyclosporine, tacrolimus, or sirolimus). Additional drugs include interferons, opioids, TNF-binding proteins, mycophenolates, and small biological agents. Examples of immunosuppressants include mesenchymal stem cells, antilymphocyte globulin (ALG) polyclonal antibody, antithymocyte globulin (ATG) polyclonal antibody, azathioprine, BAS 1L1 X 1MAB® (anti-IL-2Ra receptor antibody), cyclosporine (cyclosporine A), DACLIZUMAB® (anti-IL-2Ra receptor antibody), everolimus, mycophenolic acid, RITUXIMAB® (anti-CD20 antibody), sirolimus, tacrolimus, and / or mycophenolate mofetil.

[0188] RPE cells can be transplanted in various forms. For example, RPE cells can be introduced to the target site in the form of a single-cell suspension, attached to a matrix, or attached to a substrate or combination, such as a matrix or membrane, extracellular matrix, or biodegradable polymer. RPE cells can also be printed onto a matrix or scaffold. RPE cells can also be transplanted (co-transplanted) with other retinal cells, such as photoreceptors. The effectiveness of treatment can be assessed by various measures of visual and ocular function and structure, including, among others, best-corrected visual acuity (BCVA), retinal sensitivity to light measured by perimetry or microperimetry in dark and light-adapted conditions, full-field, multifocal, focal, or pattern electroretinography (ERG), contrast sensitivity, reading speed, color vision, clinical biomicroscopy, fundus photography, optical coherence tomography (OCT), fundus autofluorescence (FAF), infrared and multicolor imaging, fluorescein or ICG angiography, employed optics, and additional measures used to assess visual function and ocular structure.

[0189] In certain embodiments, treating, slowing, halting, or reversing retinal disease is demonstrated by recovery of visual acuity as assessed by microperimetry, wherein recovery of visual acuity as assessed by microperimetry comprises a correlation between retinal sensitivity and EZ loss in microperimetry compared to baseline, an age-matched and gender-matched control, or the fellow eye of the subject. In certain embodiments, treating, slowing, halting, or reversing retinal disease is demonstrated by recovery of visual acuity as assessed by microperimetry, wherein there is a correlation between ellipsoid zone (EZ) loss on spectral-domain optical coherence tomography (SD-OCT) and reduced retinal sensitivity on macular integrity assessment (MAIA) microperimetry. See Invest Ophthalmol Vis Sci. 2017 May 1;58(6):BIO291-BIO299.doi:10.1167 / iovs.17-21834, “Correlation Between Macular Integrity Assessment and Optical Coherence Tomography Imaging of Ellipsoid Zone in Macular Telangiectasia Type 2”; Mukherjee D. et al., which is incorporated by reference in its entirety.

[0190] In another embodiment, topographic maps of the ellipsoid region, e.g., orthogonal topographic (en face) maps, were generated from OCT volume scans, e.g., Heidelberg Spectralis OCT volume scans (15 x 10° area, 30 μm B-scan spacing) or Zeiss Cirrus HD-OCT 4000 512 x 128 cube scans, and the maps were compared with age-matched and gender-matched controls, the subject's baseline, or the subject's fellow eye to demonstrate the ability to treat, slow, halt, or reverse the progression of retinal disease. There is a correlation between EZ organization and retinal sensitivity. After administration of RPE cells, the EZ region is organized and retinal sensitivity improves. See, for example, Figures 25 and 26 at 3 months. See, e.g., Retina, 2018 Jan;38 Suppl 1:S27-S32. “Correlation Of Structural And Functional Outcome Measures In A Phase One Trial Of Ciliary Neurotrophic Factor In Type 2 Idiopathic Macular Telangiectasia,” Sallo FB, et al., which is incorporated by reference in its entirety.

[0191] In certain embodiments, treating or slowing, halting, or reversing the progression of retinal disease is demonstrated by OCT-A before and after administration compared to age-matched and gender-matched controls, the subject's baseline, or the fellow eye.

[0192] For example, spectral-domain (SD)-OCT and OCT-A imaging are used, and SD-OCT data is analyzed, e.g., using OCT EZ mapping, to obtain linear, areal, and volumetric measurements of the EZ-retinal pigment epithelium (RPE) complex across the macular cube. OCT-A retinal capillary density can be measured, e.g., using the Optovue Avanti split-spectrum amplitude decorrelation angiography algorithm. EZ-RPE parameters are compared to age- and sex-matched controls, the subject's baseline, or the fellow eye.

[0193] In one embodiment, after administration, EZ-RPE foveal mean thickness improves, EZ-RPE foveal thickness improves, and EZ-RPE central subfield volume improves. EZ-RPE thickness, area, and volume correlate with visual acuity improvement to measure treatment response. Each of these measurements inversely correlates with visual acuity. See Figures 25 and 26, where EZ volume decreases from baseline to 3 months. See, for example, the methods outlined in Invest Ophthalmol Vis Sci. 2017 Jul 1;58(9):3683-3689, "OCT Angiography and Ellipsoid Zone Mapping of Macular Telangiectasia Type 2 From the AVATAR Study," Runkle AP., et al., incorporated herein by reference in its entirety.

[0194] In one embodiment, recovery is, for example, a subjective assessment that one or more of the following are becoming more organized: the external limiting membrane, the myoid region (inner photoreceptor segment), the ellipsoid region (IS / OS junction), the photoreceptor outer segment, loss of drusen, and disappearance of reticular pseudodrusen. Recovery can also include a subjective assessment that one or more of the basic underlying layers of the retina are becoming more organized. As used herein, the basic underlying layers of the retina that are becoming more organized include one or more of the external limiting membrane, the myoid region (inner photoreceptor segment), the ellipsoid region (IS / OS junction), and the photoreceptor outer segment. As seen in Figures 25 and 26, organization is demonstrated by a decrease in the volume of structures, for example, in the EZ, e.g., comparing baseline with months 2 and 3. For example, the volume of the EZ is reduced by at least 2%, at least 5%, or at least 10%.

[0195] In one embodiment, ellipsoidal zone analysis demonstrates EZ organization by a decrease in EZ volume compared to an age-matched, gender-matched control, baseline, or fellow eye. In another embodiment, the decrease in EZ volume includes at least 2%, at least 5%, at least 7%, or at least 10%, or 1-5%, 1-10%, 1-50%, or 10-50%. In another embodiment, EZ organization is demonstrated, for example, by a decrease in the volume of EZ structures, e.g., comparing baseline to months 2 and 3. For example, EZ volume decreases by at least 2%, at least 5%, or at least 10%.

[0196] In one embodiment, the recovery includes one or more of the following: an improvement in EZ-RPE foveal mean thickness, an improvement in EZ-RPE foveal thickness, and an improvement in EZ-RPE central subfield volume. The thickness, area, and volume of EZ-RPE correlate with the improvement in visual acuity for measuring treatment response. Each of these measurements correlates inversely with visual acuity.

[0197] The RTA RPE cell therapy formulated according to the present disclosure does not require the use of GMP facilities for preparation of the final dose formulation prior to injection into the subject's eye. The RTA RPE cell therapy formulations described herein can be cryopreserved in a non-toxic freezing solution, including a final dose formulation that can be shipped directly to the clinical site. When needed, the formulation can be thawed and administered to the subject's eye without any intermediate preparation steps.

[0198] RPE cells can be generated, for example, according to the methods of Idelson M, Alper R, Obolensky A et al. (Directed differentiation of human embryonic stem cells into functional retinal pigment epithelium cells. Cell Stem Cell 2009;5:396-408), or according to Parul Choudhary et al. ("Directing Differentiation of Pluripotent Stem Cells Toward Retinal Pigment Epithelium Lineage", Stem Cells Translational Medicine, 2016), or WO2008129554, all of which are incorporated herein by reference in their entireties.

[0199] The number of viable cells that can be administered to a subject is typically at least about 50,000 to about 5x10 per dose. 6In some embodiments, the RPE cell composition comprises at least about 100,000 viable cells. In some embodiments, the RPE cell composition comprises at least about 150,000 viable cells. In some embodiments, the RPE cell composition comprises at least about 200,000 viable cells. In some embodiments, the RPE cell composition comprises at least about 250,000 viable cells. In some embodiments, the RPE cell composition comprises at least about 300,000 viable cells. In some embodiments, the RPE cell composition comprises at least about 350,000 viable cells. In some embodiments, the RPE cell composition comprises at least about 400,000 viable cells. In some embodiments, the RPE cell composition comprises at least about 450,000 viable cells. In some embodiments, the RPE cell therapy composition comprises at least about 500,000 viable cells. In some embodiments, the RPE cell composition comprises at least about 600,000, at least about 700,000, at least about 800,000, at least about 900,000, at least about 1,000,000, at least about 2,000,000, at least about 3,000,000, at least about 4,000,000, at least about 5,000,000, at least about 6,000,000, at least about 7,000,000, at least about 8,000,000, at least about 9,000,000, at least about 10,000,000, at least about 11,000,000, or at least about 12,000,000 viable cells.

[0200] In certain embodiments, the RPE cell therapy may be formulated at a cell concentration of about 100,000 cells / ml to about 1,000,000 cells / ml. In certain embodiments, the RPE cell therapy may be formulated at a cell concentration of about 1,000,000 cells / ml, about 2,000,000 cells / ml, about 3,000,000 cells / ml, about 4,000,000 cells / ml, about 5,000,000 cells / ml, 6,000,000 cells / ml, 7,000,000 cells / ml, 8,000,000 cells / ml, about 9,000,000 cells / ml, about 10,000,000 cells / ml, or about 11 The cells may be formulated at a cell concentration of about 10,000,000 cells / ml, about 12,000,000 cells / ml, 13,000,000 cells / ml, 14,000,000 cells / ml, 15,000,000 cells / ml, 16,000,000 cells / ml, about 17,000,000 cells / ml, about 18,000,000 cells / ml, about 19,000,000 cells / ml, or about 20,000,000 cells / ml.

[0201] In some embodiments, the RPE cell composition can be cryopreserved and stored at a temperature of about -4°C to about -200°C. In some embodiments, the RPE cell composition can be cryopreserved and stored at a temperature of about -20°C to about -200°C. In some embodiments, the RPE cell composition can be cryopreserved and stored at a temperature of about -70°C to about -196°C. In some embodiments, temperatures suitable for cryopreservation or cryopreservation temperatures include temperatures of about -4°C to about -200°C, or temperatures of about -20°C to about -200°C, or temperatures of -70°C to about -196°C.

[0202] In some embodiments, the cell composition is administered into the subretinal space, while in other embodiments, the cell composition is injected.

[0203] In some embodiments, the cell composition is administered as a single dose treatment.

[0204] In some embodiments, the RPE cells are administered in a therapeutically or pharmaceutically acceptable carrier or biocompatible vehicle. In some embodiments, the volume of the RPE formulation administered to a subject is about 10 μl to about 50 μl, about 20 μl to about 70 μl, about 20 μl to about 100 μl, about 25 μl to about 100 μl, about 100 μl to about 150 μl, or about 10 μl to about 200 μl. In certain embodiments, 10 μl to 200 μl of the RPE formulation can be administered two or more times. In certain embodiments, a fixed amount of the RPE formulation is administered to the subretinal space of the subject's eye. In certain embodiments, the subretinal delivery method can be transvitreal or suprachoroidal. In some embodiments, for some subjects, transvitreal or suprachoroidal subretinal delivery methods can be used to reduce the incidence of ERM. In some embodiments, a fixed amount of the RPE formulation can be injected into the subject's eye.

[0205] Subjects that can be treated include primates (including humans), dogs, cats, ungulates (e.g., horses, cattle, bovines (e.g., pigs)), birds, and other subjects. Humans and non-human animals of commercial importance (e.g., livestock and farm animals) are of particular interest. Exemplary mammals that can be treated include dogs, cats, horses, cattle, sheep, rodents, etc., and primates, particularly humans. Non-human animal models, particularly mammals, such as primates, mice, rabbits, etc., can be used for experimental studies.

[0206] The RPE cells generated as described herein can be transplanted into various target sites within the subject's eye or other locations (e.g., the brain). According to one embodiment, RPE cells are transplanted into the subretinal space of the eye, which is the usual anatomical location of RPE (between the photoreceptor outer segments and the choroid). Furthermore, depending on the cell's migration ability and / or positive paracrine effect, transplantation into other ocular compartments, including but not limited to the vitreous cavity, the inner or outer retina, the retinal periphery, and the choroid, can be considered.

[0207] Transplantation can be carried out by various techniques known in the art.The method of carrying out RPE transplantation is described in, for example, United States Patent No. 5,962,027, United States Patent No. 6,045,791 and United States Patent No. 5,941,250, and Eye Graefes Arch Clin Exp Opthalmol March 1997;235(3):149-58;Biochem Biophys Res Commun February 24,2000;268(3):842-6;Opthalmic Surg February 1991;22(2):102-8. Methods for performing corneal transplants are described, for example, in U.S. Patent No. 5,755,785 and Eye 1995;9(Pt 6 Su):6-12; Curr Opin Opthalmol August 1992;3(4):473-81; Ophthalmic Surg Lasers April 1998;29(4):305-8; Ophthalmology April 2000;107(4):719-24; and Jpn J Ophthalmol November-December 1999;43(6):502-8. When primarily utilizing paracrine action, cells can also be delivered and maintained in the eye encapsulated in a semipermeable container or a biodegradable extracellular matrix, which also reduces exposure of cells to the host immune system (Neurotech USA CNTF delivery system; PNAS March 7, 2006 vol.103(10)3896-3901).

[0208] According to some embodiments, transplantation is performed by delivering the cells into the subretinal space through a small retinal opening after pars plana vitrectomy surgery or by direct injection.

[0209] The subject may be administered a corticosteroid, such as prednisolone, methylprednisolone, or predforte, prior to or concurrently with the administration of RPE cells. According to another embodiment, the subject is not administered a corticosteroid, such as prednisolone, methylprednisolone, or predforte, prior to or concurrently with the administration of RPE cells.

[0210] Immunosuppressants can be administered to subjects before, during, and / or after treatment. Immunosuppressants can belong to the following classes: glucocorticoids, cytostatics (e.g., alkylating agents or antimetabolites), antibodies (polyclonal or monoclonal), drugs acting on immunophilins (e.g., cyclosporine, tacrolimus, or sirolimus). Additional drugs include interferons, opioids, TNF-binding proteins, mycophenolates, and small biological agents. Examples of immunosuppressants include mesenchymal stem cells, antilymphocyte globulin (ALG) polyclonal antibody, antithymocyte globulin (ATG) polyclonal antibody, azathioprine, BAS 1L1 X 1MAB® (anti-IL-2Ra receptor antibody), cyclosporine (cyclosporine A), DACLIZUMAB® (anti-IL-2Ra receptor antibody), everolimus, mycophenolic acid, RITUXIMAB® (anti-CD20 antibody), sirolimus, tacrolimus, and / or mycophenolate mofetil.

[0211] The immunosuppressant may be administered to the subject, for example, topically, intraocularly, intraretinaly, or systemically. The immunosuppressant may be administered by one or more of these methods simultaneously, or the delivery methods may be used in a staggered manner.

[0212] Alternatively, the RTA RPE cell therapy composition may be administered without the use of immunosuppressive drugs.

[0213] Antibiotics may be administered to the subject prior to, concurrently with, and / or after treatment. Examples of antibiotics include ofloxacin, gentamicin, chloramphenicol, tobrex, vigamox, or any other topical antibiotic preparation approved for use in the eye.

[0214] In some embodiments, the cell composition does not cause inflammation after administration, hi some embodiments, inflammation can be characterized by the presence of cells associated with inflammation.

[0215] AMD is a progressive, chronic disease of the central retina and a leading cause of blindness worldwide. Most blindness occurs in the later stages of the disease due to one of two processes: neovascular ("wet") AMD and geographic atrophy (GA, "dry") AMD. GA involves progressive atrophy of the retinal pigment epithelium, choriocapillaris, and photoreceptors. While dry AMD is more common (85–90% of all cases), it can progress to the "wet" form, which, if left untreated, leads to rapid and severe vision loss.

[0216] The estimated prevalence of AMD is 1 in 2,000 in the United States and other developed countries. This prevalence is expected to increase with the proportion of older people in the general population. Risk factors for the disease include both environmental and genetic factors.

[0217] The pathogenesis of this disease involves abnormalities in four functionally interrelated tissues: the retinal pigment epithelium (RPE), Bruch's membrane, the choriocapillaris, and photoreceptors. However, impaired RPE cell function is an early and critical event in the molecular pathway that leads to clinically relevant AMD changes.

[0218] Currently, there is no approved treatment for dry AMD. Preventive measures include vitamin / mineral supplements, which reduce the risk of developing wet AMD but do not affect the development of geographic atrophy (GA) progression.

[0219] Cell transplantation can be used to slow disease progression, induce RPE regeneration, and restore central vision.

[0220] Without the RPE, photoreceptor cells are inoperable. Therefore, detection of GA using imaging techniques involves identifying scotomas in the visual field. In some subjects with GA, the disease may initially progress in a unique pattern that avoids the area of ​​the retina with the highest visual acuity, such as the fovea. In these subjects, the fovea is only affected in the later stages of the disease.

[0221] Thus, the methods described herein can be used to measure the therapeutic efficacy of retinal disease therapies, such as cell therapies. In one aspect, the methods involve quantitative structural and functional assessment of the eye of a subject with a treated retinal disease.

[0222] The non-limiting list of diseases that can be measured by the described method for the effect of treatment includes retinitis pigmentosa, Leber's congenital amaurosis, hereditary or acquired macular degeneration, age-related macular degeneration (AMD), geographic atrophy (GA), Best's disease, retinal detachment, gyrate atrophy, choroideremia, pattern dystrophy and other dystrophy of RPE, Stargardt's disease, damage to RPE and retina caused by any one of light, laser, inflammation, infection, radiation, neovascularization or traumatic injury.According to certain embodiments, the disease is atrophic AMD.According to another embodiment, the disease is GA.

[0223] The FDA has approved the measurement of ocular structures as an endpoint in clinical trials for evaluating retinal disease treatments. In certain embodiments, measurements of ocular structures can be performed using fundus autofluorescence (FAF) imaging. Fundus autofluorescence allows for accurate measurement of areas of atrophy in eyes with retinal disorders. In FAF imaging, areas of atrophy appear hyperfluorescent (dark) surrounded by normal retinal tissue with mild hyperfluorescence. In the majority of subjects with GA, areas of atrophy are surrounded by a rim of intense hyperfluorescence. This hyperfluorescence is associated with areas of apoptosis and cell death. According to embodiments of the disclosed methods, measurements of hyperfluorescence can be used to confirm disease progression, particularly after treatment. Slowing or halting disease progression can be demonstrated by a reduction or disappearance of the rim of intense hyperfluorescence surrounding the area of ​​atrophy.

[0224] In certain embodiments, subjects with GA who have active lesions (i.e., atrophic areas or scars), as evidenced by the presence of a hyperfluorescent rim surrounding the atrophic area after FAF imaging, can be treated with hESC-derived RPE transplantation, for example, according to the method described in WO2016 / 108219, the entire contents of which are incorporated herein by reference, or similar methods, or new methods involving reduced immunosuppression. To measure the effect of treatment on disease progression, the lesion is first artificially divided into two halves by inserting a line across the lesion parallel to the treatment area, as generated by the FAF imaging device. The line is then moved perpendicularly to the opposite side of the treatment area until the two portions of the lesion have similar areas. The position of the line across the lesioned area of ​​the retina is kept constant throughout subsequent measurements of the subject. Half of the lesioned area is then treated with hESC-derived RPE transplantation (the treatment area), while the other half of the lesion is left untreated.

[0225] At designated times after treatment, FAF can be used to detect hyperfluorescence, particularly around the margins of the lesions, and to measure the size of the atrophic area. A reduction in the size or disappearance of the hyperfluorescent margins around the lesions, along with a reduction in the overall size of the lesion, can be used to indicate that treatment is slowing or halting disease progression. The difference in hyperfluorescence between the treated and untreated halves of the lesion can be measured and used to determine the effectiveness of treatment. Therefore, the same eye can be used as both the treatment and control subjects.

[0226] The determination that FAF can be used to demonstrate that treated areas undergo a change from hyperfluorescence to hypofluorescence, thereby indicating a slowing or halting of disease progression, is an improvement over current treatment efficacy assessment methods that use FAF. This improved procedure can be used as a surrogate for treatment efficacy in clinical trials.

[0227] In one embodiment, FAF is performed using BluePeak Blue Laser Autofluorescence (Heidelberg Engineering GmbH, Max-Jarecki-Straße 8, 69115 Heidelberg, Germany). BluePeak is a non-invasive scanning laser fundus imaging method that uses lipofuscin as an indicator to reveal metabolic stress in the retina. BluePeak images can reveal dysfunction of the RPE and photoreceptors.

[0228] In another embodiment, treatment efficacy assessment using two-dimensional imaging of fundus autofluorescence is enhanced using optical coherence tomography (OCT). OCT can be used to generate three-dimensional, high-resolution images, providing important cross-sectional information for structural assessment of retinal layers, particularly in subjects undergoing treatment for retinal diseases. Using OCT, profile images of retinal layers can be obtained before and after administration of a treatment for retinal disorders. In healthy eyes, individual layers of retinal tissue can be seen as clearly defined bands. Conversely, characteristic defects caused by, for example, AMD or GA can be seen as highly demarcated areas of degeneration of the RPE and photoreceptor layers. In many eyes with GA, OCT images can reveal wedge-shaped, hyporeflective structures that can develop between the branch membrane and outer plexiform layer. Identifying and monitoring such structures can be useful for defining the OCT boundaries of the photoreceptor layer, which is important in clinical trials of therapies aimed at preserving the viability of retinal layers in patients with AMD and GA.

[0229] Combining OCT retinal layer segmentation with metabolic mapping of fundus autofluorescence allows for a clearer view of morphological changes associated with functional changes. Using specialized software, the lesion area seen in FAF images can be quantified and tracked over time. Treatment effects, including areas of RPE regeneration overlying the lesion, can also be identified, and RPE recovery can be quantified by measuring retinal thickness.

[0230] Currently, OCT is not always the standard for assessing retinal morphology in clinical trials, but aspects of the described methods suggest that using OCT in combination with other structural and functional assessment techniques can optimize measurement of treatment efficacy and enable shorter clinical trials requiring fewer patients.

[0231] Another aspect of the methods described herein includes a functional assessment component to measure the effectiveness of treatment for retinal diseases. Currently, several functional assessment methods are available, including low-light visual acuity, contrast sensitivity assessment, reading speed assessment, microperimetry, and quality of life assessment. In one aspect, an improved method for the use of microperimetry is described.

[0232] Low-light visual acuity and contrast sensitivity measure the impact of luminance and contrast on overall visual function, but do not provide a more detailed assessment of function across specific regions of the retina. The specific location of GA or other retinal disease lesions in the macula or fovea may determine visual outcome. Therefore, a high level of detail is important for functional assessment of vision in subjects with disorders such as GA.

[0233] In microperimetry, specific areas of the retina are stimulated with a point of light, and the subject presses a button to confirm perception of the stimulus. In addition to identifying functional and non-functional areas, the stimulus intensity can also be varied to identify the relative sensitivity of specific areas of the retina. The fundus can be monitored with an infrared camera, and visual field sensitivity can be mapped onto fundus photographs and compared with images obtained with other techniques.

[0234] In some embodiments, healing of the injection site occurs within about 1 day (24 hours), 1 week, about 2 weeks, about 3 weeks, about 4 weeks, or about 5 weeks after the treatment procedure. In other embodiments, healing of the injection site occurs within about 1 day to about 30 days after administration of RPE cells. In still other embodiments, healing of the cannulated administration site occurs within 5 to about 21 days or within about 7 to about 15 days.

[0235] In some aspects, a subject treated with the RPE cells described herein exhibits an increase in BCVA after about 1 day, about 1 week, about 2 weeks, about 3 weeks, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, or about 11 months, when compared to an age-matched and gender-matched control, the subject's baseline, or a measurement in the fellow eye. In some aspects, a subject treated with the RPE cell compositions described herein exhibits an increase in BCVA after about 1 month to about 1 year of treatment with the RPE cells, when compared to an age-matched and gender-matched control, the subject's baseline, or a measurement in the fellow eye.

[0236] In some embodiments, a subject treated with the RPE cells described herein has subretinal pigmentation stabilized for about 1 month to about 24 months after administration of the treatment. In some embodiments, a subject treated with the RPE cells described herein has subretinal pigmentation stabilized for about 2 months to about 12 months, about 3 months to about 11 months, about 1 month to about 6 months, or about 4 months to about 18 months after administration of the treatment.

[0237] In some embodiments, subretinal pigmentation stabilizes about 1 month to about 24 months after administration of RPE cells to a subject. In some embodiments, subretinal pigmentation stabilizes about 2 months to about 24 months after administration of RPE cells to a subject. In some embodiments, subretinal pigmentation stabilizes about 2 months to about 12 months, about 3 months to about 11 months, about 1 month to about 6 months, or about 4 months to about 18 months after administration of RPE cells to a subject.

[0238] Subjects undergoing allogeneic cell transplantation procedures such as those described herein may develop an immune response to these cells, which may limit cell survival and functionality. Therefore, subjects may undergo systemic immunosuppressive therapy (low-dose immunosuppression based on the drug's prescribing information) before and / or after RPE cell administration, typically consisting of topical steroid therapy after vitrectomy and long-term systemic treatment.

[0239] In other embodiments, subjects receive immunosuppression for one day to three months. In other embodiments, subjects receive immunosuppression for one day to three months after RPE cell therapy. One method is to provide a tapering course of prednisolone or dexamethasone infusions four to eight times daily. Systemic (PO) tacrolimus 0.01 mg / kg / day (adjusted to achieve a blood concentration of 3-7 ng / mL) is continued up to two weeks prior to transplant and up to six weeks post-transplant at the discretion of the investigator.

[0240] Up to 2 g / day of systemic (PO) mycophenolate mofetil may be used, administered up to 2 weeks before transplant and continued for 1 year after transplant.

[0241] In one aspect, the method for improving the safety of a subject receiving treatment for dry AMD does not include the administration of immunosuppressants. In another aspect, the incidence and frequency of treatment-emergent adverse events are lower than when the subject is administered immunosuppression. [Example]

[0242] Reference is now made to the following non-limiting examples, which together with the above description illustrate certain aspects of the present disclosure.

[0243] Generally, the nomenclature used herein and the laboratory procedures utilized in this disclosure include molecular, biochemical, microbiological, and recombinant DNA techniques. Such techniques are fully explained in the literature. See, for example, "Molecular Cloning: A Laboratory Manual" by Sambrook et al. (1989); "Current Protocols in Molecular Biology" Volumes I-III Ausubel, R.M., ed. (1994); Ausubel et al., "Current Protocols in Molecular Biology", John Wiley and Sons, Baltimore, Maryland (1989); Perbal, "A Practical Guide to Molecular Cloning", John Wiley & Sons, New York (1988); Watson et al., "Recombinant DNA", Scientific American Books, New York; Birren et al. (eds) "Genome Analysis: A Laboratory Manual Series", Vols. 1-4, Cold Spring Harbor Laboratory Press, New York. New York (1998); methods described in U.S. Patent Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and 5,272,057; "Cell Biology: A Laboratory Handbook", Volumes I-III, Cellis, JE, ed. (1994); "Culture of Animal Cells - A Manual of Basic Technique" by Freshney, Wiley-Liss, NY (1994), Third Edition; "Current Protocols in Immunology", Volumes I-III, Coligan JE, ed. (1994); Stites et al.(eds), “Basic and Clinical Immunology” (8th Edition), Appleton & Lange, Norwalk, CT (1994); Mishell and Shiigi (eds), “Selected Methods in Cellular Immunology”, WHFreeman and Co., New York (1980); available immunoassays are extensively described in the patent and scientific literature, see, e.g., U.S. Patent Nos. 3,791,932; 3,839,153; 3,850,752; 3,850,578; 3,853,987; 3,867,517; 3,879,262; 3,901,654; 3,935,074; 3,984,533; 3,996,345; 4,034,074; 4,098,876; 4,879,219; 5,011,771 and 5,281,521; "Oligonucleotide Immunoassays"; “Synthesis” Gait, MJ, ed. (1984); “Nucleic Acid Hybridization” Hames, BD, and Higgins SJ, eds. (1985); “Transcription and Translation” Hames, BD, and Higgins SJ, eds. (1984); “Animal Cell Culture” Freshney, RI, ed. (1986); “Immobilized Cells and Enzymes” IRL Press, (1986); “A Practical Guide to Molecular Cloning” Perbal, B., (1984) and “Methods in Enzymology” Vol. 1-317, Academic Press; “PCR Protocols: A Guide To Methods And Applications” Academic Press, San Diego, CA (1990); Marshak et al.See, "Strategies for Protein Purification and Characterization - A Laboratory Course Manual," CSHL Press (1996); all of which are incorporated herein by reference as if fully set forth herein. Other general references are provided throughout this document. The procedures therein are believed to be well known in the art and are provided for the convenience of the reader. All information contained therein is incorporated herein by reference.

[0244] The rationale for the biological activity of RPE cells is that hESC-derived RPE cells can be safely transplanted into the subretinal space of patients with macular degeneration caused by RPE cell degeneration, replacing dead or dying RPE with functional RPE, resulting in biological benefits including a reduced growth rate of the atrophic area and a slowing or halting of associated vision loss. Transplantation with functional RPE may result in: 1) re-establishment of a functional RPE layer; 2) preservation of existing photoreceptors; 3) creation of a microenvironment that promotes the survival of existing cells and the continuation of cellular function and / or structure; and 4) an eventual slowing or reversal of disease progression, thereby preserving vision.

[0245] RPE cell transplantation, as described herein, reduces, diminishes, or halts the progression of GA and associated visual loss; preserves photoreceptor function in the transplanted area based on microperimetry and / or multifocal ERG; and demonstrates improvement or restoration of normal anatomy in affected areas as determined by changes in the ellipsoid zone (EZ), RPE survival as evidenced by optical coherence tomography (OCT), and improved retinal thickness. Furthermore, RPE transplantation preserves vision in the foveal region and improves BCVA, low-light testing, and / or reading speed.

[0246] In certain subjects (e.g., patients), the size of GA lesions is approximately 0.1 mm 2 ~approx. 500mm 2 , about 0.5mm 2 ~about 30mm 2 , about 0.5mm2 ~about 15mm 2 , about 0.1mm 2 ~about 10mm 2 , about 0.25mm 2 ~about 5mm 2 , about 5mm 2 ~approx. 50mm 2 , approximately 100 mm 2 ~approx. 500mm 2 , about 2 mm 2 ~about 25mm 2 The size of GA lesions can be measured by methods described herein or known in the art.

[0247] Exclusion criteria included patients being unable to undergo vitrectomy or having a medical history of uveitis, diabetic retinopathy, CRVO, BVO, AION, optic atrophy, ongoing treatment for active treatment of wet AMD with anti-VEGF, end-stage glaucoma, diabetic retinopathy, vascular occlusion, uveitis, Coats' disease, glaucoma, being phakic, or having the presence of moderate to severe ERM.

[0248] In one embodiment, RPE cell transplantation is administered as a single injection of 100-250K RPE, for example, in a thawed injectable formulation. Repeated administration may be required for RPE transplantation. In another embodiment, RPE transplantation is administered as a single injection of 100-250K RPE without the need for repeated administration. In certain embodiments, administration includes intravitreal subretinal injection. In other embodiments, administration includes intravitreal subretinal injection.

[0249] Example 1 Clinical Protocol The safety and tolerability of the RPE cells described herein were evaluated in a dose-escalation Phase I / IIa clinical trial in patients with advanced dry AMD with GA. Patients in Cohort 1 (Patients 1, 2, and 3, ages 74–80 years with BCVA of 20 / 200 or less) received a target therapeutic dose of 50,000 RPE cells in a volume of 100 μl. Patients in Cohort 2 (Patients 4, 5, and 6, ages 65 and 82 years, also with BCVA of 20 / 200 or less) received a target therapeutic dose of 200,000 RPE cells in a volume of 100 μl. Cohort 3 (Patients 7, 8, and 9, with BCVA of 20 / 200 or less) received 100,000 cells in a volume of 50 μl. The RPE cells discussed herein were successfully administered without any serious adverse events. Retinal imaging data showed that the administered RPE cells engrafted in patients and formed a monolayer, a characteristic of native RPE. At least for the first patient, RPE cells remained present after one year. The second patient showed similar results at six months. An additional cohort of subjects will use higher doses of 200,000 to 500,000 RPE cells.

[0250] Data from Cohort 1 showed stable visual acuity and FAF readings, indicating biological activity in patients who completed readings at 9 and 12 months. Furthermore, this early data suggests that RPE cells transplanted into patients will engraft and survive for at least one year, potentially longer. There are also some early signs of biological activity.

[0251] The study design involves a single-center Phase I / IIa study of patients with advanced dry AMD and geographic atrophy (GA) divided into four cohorts: the first three cohorts, each consisting of three legally blind individuals with best-corrected visual acuity of 20 / 200 or less, were divided into cohort 1, 50x10 3 cells, 200x10 for cohort 2 and cohort 3 3Patients received a single subretinal injection of RPE cells using increasing doses of cells. The fourth cohort included nine patients with best-corrected visual acuity of 20 / 64 to approximately 20 / 400, 20 / 70 to approximately 20 / 400, or approximately 20 / 64 or less, and received a single subretinal injection of 200,000 to 500,000 RPE cells.

[0252] After vitrectomy, cells are delivered to the subretinal space in the macula via a cannula through a small retinotomy. A total volume of approximately 50-250 μl of cell suspension is injected into the area at risk for GA expansion.

[0253] In addition to surgical procedures, patients may receive mild immunosuppressive and antibiotic treatment, including: 1. Routine topical steroid and antibiotic treatment after vitrectomy: topical steroid therapy (Predforte eye drops, tapered 4-8 times daily) and topical antibiotic eye drops (Ofloxacin or equivalent 4 times daily) for 6 weeks. 2. Systemic (PO) tacrolimus 0.01 mg / kg / day (dose adjusted to achieve a blood concentration of 3-7 ng / ml) beginning 1 week before transplant and continuing until 6 weeks after transplant. 3. Systemic (PO) mycophenolate mofetil, 2gr / day total, administered 2 weeks prior to transplant and continued for 1 year after transplant.

[0254] This enhanced protocol could potentially shorten the duration of immunosuppression from 12 months to 3 months, which is important for patients. We plan to administer RPE cells without immunosuppression, which we hope will provide similar or improved efficacy with improved safety.

[0255] Patients will be evaluated at pre-scheduled intervals for 12 months after cell administration. Post-study follow-up will occur at 15 months, 2, 3, 4, and 5 years after surgery.

[0256] Patient inclusion criteria included the following: age 55 years and older; diagnosis of dry (non-neovascular) age-related macular degeneration in both eyes; and size of 0.5 disc area (1.25 mm) in the study eye. 2 and up to 17mm 2 ophthalmoscopic findings of dry AMD with geographic atrophy in the macula exceeding 0.5 disc area in the fellow eye; best-corrected central visual acuity of the study eye by ETDRS visual acuity test of 20 / 200 or less in cohorts 1-3 and 20 / 64 or less in cohort 4; visual acuity of the non-operated eye must be greater than or equal to that of the operated eye; patients in good enough health to participate in all study-related procedures and to complete the study (medical records); ability to undergo vitreoretinal surgery under monitored anesthetic control; normal blood counts, blood chemistry, coagulation, and urinalysis; negative for HIV, HBC, and HCV, CMV IgM, and EBV Negative for IgM; no current or history of malignancy (except successfully treated basal / squamous cell carcinoma of the skin) based on age-matched screening tests (at the discretion of the investigator); Patients are permitted to discontinue aspirin, aspirin-containing products, and any other coagulation-modulating medications for 7 days prior to surgery; are willing to refrain from all future blood and tissue donations; and are able to understand and willing to sign informed consent.

[0257] Patient exclusion criteria included the following factors: evidence of neovascular AMD by medical history and by clinical examination, fluorescein angiography (FA), or optical coherence tomography (OCT) at baseline in either eye; history or presence of diabetic retinopathy, vascular occlusion, uveitis, Coats' disease, glaucoma, cataract, or medial optic opacity that interferes with posterior pole visualization, or any significant ocular disease other than AMD that has impaired or may impair vision in the study eye and may confound analysis of the primary outcome; History of retinal detachment repair in the study eye; axial myopia greater than -6 diopters; ophthalmic surgery in the study eye within the past 3 months; history of cognitive impairment or dementia; contraindication to systemic immunosuppression; history of a disease other than AMD associated with choroidal neovascularization in the study eye (e.g., pathological myopia or presumed ocular histoplasmosis); active or history of the following diseases: cancer, renal disease, diabetes, myocardial infarction within the past 12 months, immunodeficiency; female gender; pregnancy or lactation; current participation in another clinical trial; previous participation (within 6 months) in a clinical trial of a systemically or ocularly administered drug.

[0258] Efficacy may be measured by graft survival and by examination of the rate of progression of GA, retinal sensitivity in the engrafted area, extent and depth of central scotoma, and change in visual acuity.

[0259] Adverse event (AE) means any untoward medical occurrence, unintended illness or injury, or adverse clinical sign (including abnormal laboratory findings) in a subject, user, or other person, whether or not related to an investigational medical treatment.

[0260] Serious Adverse Event (SAE) means an adverse event that resulted in death, injury, or permanent impairment of a body structure or function, caused a serious deterioration in the health status of a subject, resulted in life-threatening illness or injury or permanent impairment of a body structure or function, or inpatient hospitalization or prolongation of an existing hospitalization, or medical or surgical intervention to prevent a life-threatening illness, caused fetal distress, fetal death, or congenital abnormality or birth defect.

[0261] No treatment-related SAEs were reported in this study.

[0262] In this example, the eye selected for RPE administration is the one with the poorest visual function. Surgery may be performed with a retrobulbar or peribulbar anesthetic block with monitored intravenous sedation or with general anesthesia, at the surgeon's discretion and in discussion with the patient. The eye undergoing surgery is prepped according to institutional protocol and aseptically draped. After placement of a lid retractor, a standard three-port vitrectomy is performed. This may include placement of a 23G infusion cannula and two 23G ports. After visual inspection of the infusion cannula within the vitreous cavity, the infusion line is opened, ensuring that the ocular structure is maintained throughout the procedure. A central vitrectomy is then performed carefully with standard 23G instruments, followed by peeling of the posterior vitreous surface, allowing unobstructed access to the posterior pole.

[0263] In this example, RPE cells are introduced into the subretinal space by penetrating the retina at a predetermined site within the posterior pole, preferably in an area close to the border of the GA while still being relatively preserved. Avoiding blood vessels, the cells are delivered to the subretinal space via the formation of a small bleb in a volume of 50-150 μl.

[0264] The delivery system may consist of a 1 mL syringe connected to a Peregrine 25G / 41G flexible retinal cannula via a 10 cm extension tube.

[0265] Cells refluxed into the vitreous cavity can be removed, and fluid-air exchange can occur. Before removing the infusion cannula, a careful examination can be performed to ensure that no iatrogenic retinal tears or breaks have occurred. The infusion cannula can then be removed. Subconjunctival antibiotics and steroids can be administered. The eye may be covered with a patch and plastic shield. The surgical administration procedure can be recorded.

[0266] In this example, a low dose of 50,000 cells / 50-150 µL or 50,000 cells / 100 µL, a medium dose of 200,000 cells / 100 µL (or 100,000 cells / 50 µL), and a high dose of 500,000 cells / 50-100 µL were used. The dose selection was based on the safety of the maximum feasible dose tested in preclinical studies and the human equivalent dose calculated based on the size of the eye and bleb.

[0267] The treatments provided herein include suspensions of therapeutic RPE cells delivered subretinally. These are highly purified, differentiated human pluripotent stem cells that are also "xeno-free," meaning that no animal products are used at any point in the derivation and production process. (See, e.g., Idelson M, et al. 2009. "Directed differentiation of human embryonic stem cells into functional retinal pigment epithelium cells." Cell Stem Cell Oct 2, 5(4):396-408 and Tannenbaum SE, et al. 2012. "Derivation of xeno-free and GMP-grade human embryonic stem cell platforms for future clinical applications." PLoS One. 7(6):e35325, both of which are incorporated herein by reference in their entireties.)

[0268] RPE cells administered in a clinical-stage trial targeting a major unmet medical need in dry AMD. Age-related macular degeneration, or AMD, is the leading cause of blindness in people over the age of 60. The number of people affected by dry AMD is estimated to be nine times greater than the number of people affected by wet AMD. However, there are currently no approved products for dry AMD.

[0269] Example 2 RPE cell growth and survival in two primary subjects The efficacy of hESC-derived RPE cell transplantation for treating dry AMD and GA was measured in two primary subjects using embodiments of the methods described herein. The two subjects were treated with hESC-derived RPE transplantation according to the methods described in WO2016 / 108219, similar methods, or novel methods involving reduced immunosuppression. New RPE growth was demonstrated by measuring increases in retinal thickness using optical coherence tomography (OCT). Data were also collected showing that the transplanted cells could survive under the retina for six months after transplantation. Figure 1 shows a schematic diagram of an example of a cell-based therapy used to replace, support, or both replace and support dysfunctional and degenerating RPE in dry AMD with GA.

[0270] The size of the lesions in these two early subjects was measured using FAF, and an improved method was used to measure the size of the hyperfluorescent rim surrounding the lesions to determine whether the transplanted cells had an effect on disease progression.

[0271] Data collected from these two subjects showed that hyperfluorescence decreased or disappeared in half of the lesions closest to the treatment area, demonstrating halted disease progression.

[0272] Example 3 Safety and Efficacy Results from Cohorts 1 and 2 of the Clinical Trial We present safety and imaging data from patients in Cohort 1 (patients 1, 2, and 3) who underwent subretinal transplantation of 50,000 RPE cells in suspension, and Cohort 2 (patients 4, 5, and 6) who underwent subretinal transplantation of 200,000 RPE cells in suspension.

[0273] The patients were elderly and had significant visual loss and large areas of clinically significant GA. The demographics and baseline characteristics of the subjects are shown in Table 1.

[0274] Table 1. Subject age and AMD characteristics at baseline TIFF2025161869000002.tif34128* One patient could only count fingers.

[0275] For cohorts 1 and 2, RPE cell transplantation was performed via subretinal injection after 23G vitrectomy under local anesthesia. For example, injections can be performed according to methods such as those described in WO2016 / 108219, the entire contents of which are incorporated herein by reference. Patients in cohorts 1 and 2 were administered systemic immunosuppression from one week before transplantation until one year after transplantation. However, methods without immunosuppression can also be used. Systemic and ocular safety were carefully monitored. Retinal function and structure were assessed using various techniques, including BCVA, color and fundus autofluorescence (FAF) imaging, and optical coherence tomography (OCT).

[0276] Figure 2A presents best-corrected visual acuity (BCVA) for the treated eyes of Cohort 1 (Patients (Pts) 1, 2, and 3). As shown, BCVA did not decline in the treated eyes of Patients 1, 2, or 3. Patient 2 showed a marked improvement, which may be related in part to the resolution of vitreous and posterior capsule opacification that occurred during surgery. The BCVA of the fellow eye is shown in Figure 2B and remained stable over the 1-year period examined.

[0277] As shown in Figure 2C-F, BCVA remained stable and did not decline in the treated eyes of Cohort 2 (patients 4, 5, and 6) and remained stable in the fellow eyes. Treated eyes of individual patients are shown in Figure 2C and Figure 2E.

[0278] The retina contains the neurosensory tissue of the eye, which converts visual images into electrical impulses that the brain interprets. Fundus photography, which records the retina, was also used to monitor disease progression and treatment efficacy. Color fundus images of Cohort 1 at preoperative (preoperative) and intraoperative (intraoperative) time points are shown in Figure 3. The border of the subretinal bleb (treatment area) that developed after injection of the therapeutic RPE cell suspension is highlighted by arrows in the intraoperative images. The surgery was uneventful, and the subretinal fluid was absorbed within 48 hours. As shown in Figure 3, patients in Cohort 1 developed large areas of GA, and images obtained intraoperatively reveal the correct placement of the transplanted cells.

[0279] Color fundus images of Cohort 1 preoperatively and at 2 months are shown and compared in Figure 4. Postoperatively, patients 1 and 2 demonstrate areas of subretinal pigmentation that developed beneath the subretinal bleb over the first 2–3 months. After the first 2–3 months, the subretinal pigmentation began to stabilize, as shown in Figure 5.

[0280] Referring to Figure 6, blue autofluorescence images from Patient 1 are provided preoperatively and at 1 day, 1 week, 2 months, 4.5 months, and 9 months postoperatively (after surgery). Blue fundus autofluorescence (FAF) imaging of the treated subject helps illustrate the large area of ​​GA and the lower limit of the retina (depicted by the dotted line) treated with RPE cells. These FAF images also show evidence of transplanted RPE cells, indicated by the black arrows, at the indicated time points.

[0281] Blue autofluorescence images from patient 2 preoperatively and at 1 day, 1 week, 2 months, 6 months, and 9 months postoperatively are seen in Figure 7. Blue autofluorescence images from patient 3 preoperatively and at 1 day, 1 week, 2 months, 7 months, and 9 months postoperatively are seen in Figure 8.

[0282] Subretinal hypofluorescent and hyperfluorescent spots developed in the inferior region of the subretinal bleb in patients 1 and 2 over the first 2–3 months and then stabilized. Figure 6 and Figure 7 reveal a gradual increase in cell number, pigment epithelial (PE) development, and surface area covered by RPE cells, indicated by the black arrow in the upper right corner of the postoperative image in Figure 6.

[0283] Figure 9 shows color images of patient 4 (Cohort 2) who received a 200,000 RPE cell suspension at the time of surgery (day 0), FAF and color images at postoperative day 1, and color images at 2, 3, 4, and 6 months after surgery. Subretinal pigmentation was observed at the border of the bleb area for up to 6 months. As shown in the images, gravity allowed the cells to settle, localizing pigmentation to the bleb border.

[0284] Figure 10 shows color and corresponding FAF images at 0 days, 1 month, 2 months, 3 months, and 6 months post-operatively for patient 5 (Cohort 2), who also received the 200,000 RPE cell suspension. As shown in Figure 10, treatment was well tolerated, with stable pigmentation increases through 6 months.

[0285] Figure 11 shows healing of the injection site. As shown, the subretinal fluid was rapidly absorbed (in less than 48 hours), and OCT images show healing of the cannula retinal penetration site (arrow) within 2 weeks. In some cases, a thin epiretinal membrane (ERM) developed.

[0286] OCT scans can be used to analyze changes in the transition zone after treatment with RPE cells. In retinal degenerative diseases, a transition zone occurs between a relatively normal retina containing healthy photoreceptors and a severely affected retina (e.g., GA lesions, pre-GA lesions) with extreme photoreceptor atrophy. OCT scans were used to analyze the transition zone in patients from Cohort 1 (patients 1, 2, and 3) and Cohort 2 (patients 4 and 5).

[0287] OCT scans were obtained for patient 1 preoperatively and at 1 week, 1 month, and 1 year postoperatively and are shown in Figure 12. OCT scans of patient 2 preoperatively and at 1 and 9 months postoperatively are shown in Figure 13. Figure 14 shows OCT scans of patient 3 in cohort 1 preoperatively and at 3 and 9 months postoperatively. Figure 15 shows OCT and infrared OCT scans of patient 4 in cohort 2 preoperatively and at 1 month postoperatively. Figure 15 shows the FAF (column 1), infrared OCT scan (column 2), and OCT scan (column 3) of patient 4 in cohort 2 preoperatively and at 1 month postoperatively.

[0288] Postoperative OCT scans in Figures 12, 13, and 15 show irregular reflectivity (yellow arrows) in the subretinal space of treated areas, including areas that were atrophic at baseline (green arrow in Figure 12). This irregular reflectivity may indicate the presence of new RPE cells within the subretinal space. Images from subjects in Cohort 2 suggest subretinal layering of transplanted hESC-RPE cells. Fundus autofluorescence (FAF), infrared SLO (IR SLO), and spectral-domain OCT (SD-OCT) images taken at baseline, 1 month, and 9 months follow-up are presented. White vertical lines indicate the boundaries of the cartographic regions in the IR SLO and OCT images. Green lines represent the SD-OCT scans in the right column. The dotted yellow line represents the lower limit of the retina treated with RPE cells. This line was taken from a fundus image immediately after surgery and superimposed on the other image modalities.

[0289] Referring to the fundus images in Figure 15, hypofluorescent spots are observed at the bottom of the treated bleb over time, revealing a decrease in disease progression. Pigmentation is also seen at the bleb border. In the infrared OCT images in Figure 15 (center column), pigment cells are observed obscuring the top of the GA (the red line indicates the GA border) one month after surgery. This reveals that the cells have the ability to migrate and uniformly cover the top of the GA, rather than remaining localized at the edge of the bleb. Because infrared OCT has the ability to penetrate several layers of the retina, cells, normal tissue, and scars can all be observed.

[0290] In the last column of Figure 15, preoperative OCT images show areas of GA with exposed RPE cells. However, OCT images taken at 1 and 9 months postoperatively demonstrate RPE cell engraftment (yellow arrows). At 1 month, a uniform monolayer of RPE cells covers the defect shown in the preoperative image, demonstrating restoration of pigment epithelium and retinal thickness. At 9 months, the pigment epithelium is as thick as the normal cellular areas shown to the left and right of the GA border. Additionally, some areas demonstrated structural improvement of the ellipsoid zone (EZ). The EZ is a critical region of the retina related to visual function, where RPE cells contact photoreceptors and where the visual process begins.

[0291] Figure 16 shows OCT scans of patient 5 (200,000 RPE cell suspension dose) in cohort 2 at baseline, 1 week, 2 weeks, 1 month, 2 months, 3 months, and 6 months post-surgery. The lack of edema or cysts observed in patient 5 (which would be present if an autoimmune response was present) indicated that the treatment was well tolerated and comparable results could be achieved with non-immunosuppressant approaches.

[0292] Subretinal transplantation was well tolerated in all patients, and accumulated data from cohorts 1 and 2, which received 50,000 or 200,000 cells in suspension, with up to 15 months of follow-up, showed no serious systemic or unexpected ocular adverse effects. After transplantation of hESC-derived RPE into the subretinal space of patients with advanced dry AMD, SD-OCT images demonstrate healing of the cannula retinal penetration site within 2 weeks. BCVA remained stable, and subretinal pigmentation, correlating with irregular subretinal hyperreflectance on OCT imaging, was evident in the majority of patients, demonstrating the presence of new RPE cells in the subretinal space. These results provide a framework for structural and functional evaluation in future cohorts treated with higher cell doses.

[0293] Example 4 Subretinal transplantation of hESC-RPE cells in pig eyes Human embryonic stem cell-derived RPE cells (hESC-RPE cells) obtained by the above method were subretinal transplanted into pig eyes to further analyze safety and cell survival. OCT scans performed 3 months postoperatively (Figure 17) demonstrate irregular reflectivity in the subretinal space (yellow arrow in the upper right image), similar to that seen in treated patients in Cohorts 1 and 2 (see Figures 12-16). This irregular reflectivity can be compared to the area beyond the bleb boundary (pink arrow), where the reflectivity of this layer is uniform.

[0294] Histological analysis was also performed. Immunohistochemistry (ICH) using the human-specific marker TRA-1-85 was performed. The TRA-1-85 antigen is a cell surface determinant expressed by nearly all human cell types and is used in somatic cell hybridization studies to identify tissues of human origin. Histological examination revealed stratification of the transplanted human cells under the retina (shown in red in Figure 17). These results demonstrate that the transplanted RPE cells were present in areas that showed irregular reflectivity on OCT scans several months after administration and were distinguishable from native porcine RPE.

[0295] Example 5 Tumorigenicity, engraftment, and survival of hESC-derived RPE cells in NOD-SCID mice The tumorigenicity, engraftment, and survival of hESC-derived RPE cells were tested in NOD-SCID mice for up to 9 months. In this assay, 100,000 hESC-derived RPE cells in suspension were injected into the subretinal space of NOD-SCID mice. hESC-derived RPE cells were prepared as described above. The positive control group received subretinal injections of hESC fragments. The vehicle control group received BSS Plus.

[0296] As shown in Table 2, no teratomas or human tumors were observed in 142 mice subretinally injected with hESC-derived RPE at a dose of 100,000 cells. Surprisingly, no teratomas were observed in the group of mice subretinally injected with hESC-derived RPE, where the hESC-derived RPE cell suspension contained up to 10% hESCs, which is 1,000-fold higher than when injected into human subjects. Fewer than 5% of mice had rare hESC-RPE outgrowths observed at 9 months. As shown in Table 2, mice injected with hESC-derived RPE cells at a dose of 100,000 cells in suspension and similarly prepared hESCs demonstrated a reduced potential for subretinally formed teratomas (less than 15%). As shown in Figure 18, teratomas were observed in the majority (54.5%–80%) of positive control animals injected with hESC fragments (arrows indicate benign teratomas).

[0297] Table 2. Tumorigenicity and viability of hESCs, hESC fragments, and hESC-derived RPE at 9 months after subretinal injection. TIFF2025161869000003.tif29141

[0298] In mice injected with hESC-derived RPE cells at a dose of 100,000 cells in suspension, long-term consistent engraftment and survival were measured using subretinal space histology after 9 months. As shown in Table 2, 89.5%–96.4% of injected mice had pigmented cells in the subretinal space, and 83%–93% had RPE. Figure 19 shows hESC-derived RPE in the subretinal space of a mouse injected with 100,000 hESC-derived RPE cells in suspension (arrows point to hESC-derived RPE in the subretinal space). Figure 20 shows images of HuNu+PMEL17+ stained cells, revealing the presence of hESC-derived RPE cells in the subretinal space after 9 months of injection of 100,000 hESC-derived RPE cells. Human cell nuclei were stained with an anti-human nuclear antibody, and mouse nuclei were counterstained with DAPI.

[0299] NOD-SCID mice (male and female) administered subretinally with doses of up to 100,000 hESC-derived RPE cells demonstrated long-term, consistent hESC-derived RPE cell survival in the subretinal space over the 9-month study period, with no product-related teratomas / tumors / abnormalities. Administration of hESC-derived RPE containing up to 10% hESC impurities did not result in teratoma formation.

[0300] Additionally, Figure 21 demonstrates the engraftment and survival of hESC-derived RPE in the retinas of three animal species using staining to indicate the presence of human cells: RCS rats 19 weeks after transplantation of hESC-derived RPE, non-SCID mice 9 months after transplantation of hESC-derived RPE, and pig retinas 3 months after transplantation of hESC-derived RPE. The arrow in the RCS rat retina image indicates the location of anti-GFP staining and RPE cell engraftment. The arrow in the non-SCID mouse retina image indicates anti-human nuclear staining. The arrow in the pig retina image indicates staining for the human-specific marker TRA-1-85.

[0301] Example 6 Safety and efficacy results for patient 8 in cohort 3 of the clinical trial Patient 8 received a subretinal injection of 100,000 hESC-derived RPE cells in 50 μL as described above. Figure 22A is a blue autofluorescence image taken before surgery, showing a baseline image of the GA (dark area), the outline of the future bleb border (dotted line), and the exact transplantation location (asterisk). Figure 22B is a color fundus image taken before surgery, showing a baseline image of the GA (dark area), the outline of the future bleb border (dotted line), and the exact transplantation location (asterisk). Figure 22C is a color image of the transplanted bleb at the time of surgery.

[0302] Figure 23 shows a color fundus image at 1 month. Slight subretinal hypofluorescence is seen above the bleb at 1 month.

[0303] Figures 24A, 24B, and 24C are blue autofluorescence images taken at 1, 2, and 3 months, respectively. As shown in the images, hypofluorescent spots are seen beneath the treated blebs over time, demonstrating a slowing of disease progression. Hyperpigmented patches are also observed within the bleb area.

[0304] Figures 25, 26, and 27 show infrared and corresponding OCT images of different cross sections of the transition zone of Patient 8 at baseline (pre-surgery), 1 month, 2 months, and 3 months. The vertical arrows in the baseline and 1 month OCT images in Figures 25 and 26 indicate some of the drusen present at these time points. A significant reduction in these drusen was observed 2 and 3 months after treatment with the hESC-derived RPE cell composition. Furthermore, the OCT images taken at 3 months show the recovery and re-establishment of the ellipsoid region, as exemplified by the area highlighted by the horizontal arrow. These images demonstrate the recovery of the ellipsoid region by ellipsoid region analysis. Ellipsoid region analysis involves, for example, visual analysis of the ellipsoid region. Ellipsoid region analysis involves visual analysis of the ellipsoid region, comparing the subject's ellipsoid region with that of an age- and gender-matched control, the subject's baseline, or the subject's fellow eye.

[0305] Recovery is indicated by subjective assessment of the inner and outer segments, including, for example, the ellipsoid zone (EZ)—the inner segment / outer segment (IS / OS) junction. Recovery is indicated by restoration of normal structure (see bottom images in Figures 25, 26, and 27). Recovery is indicated by restoration of normal structure, for example, when compared to age-matched and gender-matched controls, the subject's baseline, or the subject's fellow eye. Restoration of normal structure indicates potential restoration of vision. For example, recovery is indicated by subjective assessment indicating the onset of visibility of one or more of, for example, the outer limiting membrane, the myoid zone (photoreceptor inner segment), the ellipsoid zone (IS / OS junction), the photoreceptor outer segment, and loss of drusen. In some subjects, reticular pseudodrusen disappear. In some embodiments, recovery is demonstrated by organization of the basic basal layers of the retina, organization of 2-6 of the 12-14 layers of the retina.

[0306] Recovery is, for example, a subjective assessment that one or more of the following are becoming more organized: the external limiting membrane, the myoid region (inner photoreceptor segment), the ellipsoid region (IS / OS junction), the outer photoreceptor segment, loss of drusen, and disappearance of reticular pseudodrusen. Recovery can also include a subjective assessment that one or more of the basic underlying layers of the retina are becoming more organized. As used herein, the basic underlying layers of the retina that are becoming more organized include one or more of the external limiting membrane, the myoid region (inner photoreceptor segment), the ellipsoid region (IS / OS junction), and the outer photoreceptor segment.

[0307] The uniform brownish color seen in FAF images of cohorts 1-3 is consistent with pigment cells, as opposed to the dark color seen when pigment dispersion occurs in response to RPE damage. In at least four patients, pigmentary changes were observed within the bleb area, both outside and inside the border of GA. These pigmentation changes and areas of autofluorescence seen in FAF images correspond to the findings in OCT images, where new subretinal material appears as a fine layer resembling RPE in areas where RPE has been lost in these patients. These results demonstrate that transplanted hESC-derived RPE cells have the ability to survive and integrate into the host retina.

[0308] Surgical safety assessments may include nonhealing retinal detachment, proliferative vitreoretinopathy (PVR), subretinal, retinal, or intravitreal hemorrhage, and damage to the still relatively healthy retina at the surgical site. However, these events were not observed in any of the cohorts in this study. Bleb formation did not cause damage to either the RPE or the neurosensory layer. Similarly, no retinal breaks or ruptures occurred. The absence of retinal breaks is noteworthy because the retina overlying GA is thinner, making the risk of retinal breaks nonnegligible.

[0309] Findings using various imaging modalities suggest the presence of cells in the subretinal space in human subjects, an observation supported by animal data from mouse, rat, and pig models tested using hESC-derived RPE cells. The surgical procedure was well tolerated, with SD-OCT images demonstrating absorption of subretinal fluid within the bleb within 48 hours and healing of the cannula retinal penetration site within several weeks. BCVA remained stable in the treated eyes of these advanced patients. Subretinal pigmentation, correlating with irregular subretinal hyperreflectance on OCT, was evident in the majority of patients (5 / 6), suggesting the presence of cells in the subretinal space.

[0310] Future cohorts will have additional methods to proactively assess visual change, and based on these results, a variety of additional objective and subjective assessments, such as microperimetry, low-light visual acuity, and reading speed, will be incorporated to determine potential efficacy.

[0311] Example 7 Subretinal RPE transplantation procedure The surgical procedure is based on conventional pars plana vitrectomy (PPV) followed by subretinal injection of a cell suspension of RPE cells.

[0312] Preoperative stage Pupil dilation in the operated eye Cyclopentolate hydrochloride 1% (3 times every 5 minutes) Phenylephrine hydrochloride 2.5% (3 times every 5 minutes) Tropicamide 1% (3 doses every 5 minutes) or Follow the facility's standard surgical procedures

[0313] anesthesia Retrobulbar or sub-Tenon block General anesthesia can be performed according to surgeon standards Light sedation may be administered according to the surgeon's standards Peribulbar or retrobulbar anesthetic administered according to the standard of care (a commonly used combination consists of lidocaine 2% and bupivacaine 0.75%)

[0314] Cleaning Povidone-iodine solution or follow your facility's standard surgical procedures

[0315] Vitrectomy ·Perform a standard three-port pars plana vitrectomy. DORC is compatible with 23G. 23G trocar system. Combination 23G / 25G trocar system A fourth trocar may be added for "chandelier" type lighting. Stain the vitreous using triamcinolone (ophthalmic) 40 mg / ml (4% concentration) to ensure complete separation of the posterior vitreous: Inject undiluted triamcinolone acetonide (0.1–0.3 ml) into the vitreous cavity via a soft-tip cannula toward the area to be visualized (e.g., the optic nerve head and posterior pole). ·Removal of preoperatively identified vitreous traction (e.g., vitreomacular traction, significant preretinal membranes). · Optionally, use intraoperative OCT (if available) to confirm that complete separation of the posterior vitreous plane has been achieved.

[0316] Preparation of the delivery device (DD) Carefully mix the RPE cells 2-3 times by filling the syringe with the cell suspension and expelling it into the vial. Fill the syringe with 0.35 mL of RPE cell suspension. Remove the 18G needle while holding the syringe upright and expel all air and bubbles by depressing the plunger and tapping the syringe. Connect the syringe to the extension tubing of the DORC delivery device Fill a DORC extendable 41G subretinal injection needle with the RPE cell suspension until a droplet appears at the tip of the cannula. Pull back slightly on the plunger (or pump if using a microdose) to allow a small amount of air into the tip (this helps identify the tip as being in the subretinal space during the initial air injection, helps expand the subretinal space with air before cell injection, and reduces the risk of cells refluxing into the vitreous cavity during cell injection). Start the timer to record the time the cells are held in the device. The time from the preparation of the DD to the start of the transplant should not exceed 2 minutes. · Turn the timer on when the DD is ready and off when the transplant begins Once the DD is attached and assembled, keep inverting / rotating the DD and do not let it lie flat / still, as cells may settle inside the syringe and tubing. Immediately begin cell transplantation within 2 minutes of attaching the DD. If more than 2 minutes have passed since the DD was assembled, discard the installed DD and prepare a new DD.

[0317] Transplantation of RPE cells Identify pre-selected injection sites based on patient images. The injection site should be located at least one papillary diameter away from the edge of the geographic atrophy (GA) lesion, superior or temporal to the GA lesion, or on the surrounding healthy tissue on or close to the GA lesion. Insert the cannula through the port, place the tip at the pre-planned retinal location for injection, and carefully penetrate the retina. Begin injecting RPE cells slowly into the subretinal space, ensuring that the tip of the cannula is in the subretinal space. Once the bleb begins to form, gently advance the tip of the cannula into the subretinal space (to avoid backflow of RPE cells from the subretinal space) and continue gentle injection until the desired amount of RPE cells is delivered into the subretinal space. If the bleb appears to be expanding in an undesirable direction, discontinue the injection and consider transplanting the remaining RPE cells to a different location.

[0318] If reflux is observed during transplantation, the surgeon should immediately stop the RPE cell injection and then perform a complete vitrectomy to ensure that most reflux cells are removed from the vitreous.

[0319] If reflux is not observed during implantation, review the videotape to confirm that reflux did not occur before completing the procedure. If reflux is observed during review, a complete vitrectomy should be performed to ensure that most reflux cells in the vitreous are removed.

[0320] If an additional bleb is required (for reasons explained above), the location of the new bleb may be at or near the location of the original bleb into which the RPE cells were transplanted. Make sure the entire bleb is visible Gently deliver 50 μL of the RPE cell suspension into the subretinal space. Gently and slowly remove the cannula. Record the time the surgery was completed

[0321] Post-surgery At the end of the procedure, apply the following: Sub-Tenon cefuroxime 0.1cc (10mg / ml) or equivalent antibiotic, and / or Maxitrol eye ointment (3.5 mg neomycin sulfate per gram, 1 mg polymyxin b sulfate 10,000 [USP], and 1 mg dexamethasone per gram) should be administered once postoperatively.

[0322] Factors that may affect the outcome include, for example, the retinal area selected, the number of attempts to create the bleb (more attempts result in less optimal results), complications, the degree of reflux (none, mild, moderate, or severe), the use of triamcinolone, the vitreous irrigation performed, whether reflux occurred, whether vitreous pigment cells were removed, and any concomitant medications administered.

[0323] Eckardt, C., Tran's conjunctival suture less 23-gauge vitrectomy.Retina, 2005.25(2):p.208-11.

[0324] Fujii, GY, et al., A new 25-gauge instrument system for trans-conjunctival sutureless vitrectomy surgery. Ophthalmology, 2002.109(10):p.1807-12; discussion 1813.

[0325] (Table 3) Overview of subjects 1 to 9 TIFF2025161869000004.tif119128 * ambiguous HRA = Heidelberg retinal angiography; OCT = optical coherence tomography; FAF = fundus autofluorescence; CFP = color fundus (retinal) photography

[0326] Subjects 1-9 have demonstrated no treatment-related systemic SAEs to date, although two unrelated SAEs occurred in two subjects; no unexpected ocular AEs have been observed; expected AEs include surgery-related conjunctival hemorrhage, worsening cataract, and epiretinal membrane formation (ERM); new ERM or worsening ERM has been observed (8 / 9); there has been no retinal edema, suggesting an immune response against RPE cells.

[0327] Subjects 1-8 show that at least 75% of subjects have RPE cells 2-24 months after treatment. At the time this data was generated, it was too early to see any signs of cells in subject 9.

[0328] Although the description herein contains many details, these should not be construed as limiting the scope of the disclosure, but merely as providing an illustration of some of the presently preferred embodiments, and therefore, it will be understood that the scope of the present disclosure fully encompasses other embodiments that may become apparent to those skilled in the art.

[0329] In the claims, reference to an element in the singular is not intended to mean "only one" but rather "one or more" unless expressly stated otherwise. All structural, chemical, and functional equivalents to the elements of the disclosed embodiments known to those of ordinary skill in the art are expressly incorporated herein by reference and are intended to be encompassed by the claims. Furthermore, no element, component, or method step of the present disclosure is intended to be dedicated to the public, regardless of whether that element, component, or method step is expressly recited in a claim. No claim element herein should be construed as a "means-plus-function" element unless the element is expressly recited using the phrase "means for." No claim element herein should be construed as a "step-plus-function" element unless the element is expressly recited using the phrase "steps for."

Claims

1. 1. A pharmaceutical composition for treating or slowing the progression of a retinal disease or disorder in a subject, comprising a therapeutically effective amount of retinal pigment epithelial (RPE) cells, wherein the therapeutically effective amount of RPE cells is between about 50,000 and 5,000,000 cells, and wherein the composition is formulated for transplantation into an area overlying a geographic atrophy (GA) lesion or an area overlying the fovea in the subject.

2. 10. The pharmaceutical composition of claim 1, wherein the subject has a best corrected visual acuity (BCVA) of 20 / 64 or less, 20 / 70 or less, or from about 20 / 64 to about 20 / 400.

3. 3. The pharmaceutical composition of claim 1 or 2, wherein administration of a therapeutically effective amount of RPE cells results in an increase in pigmentation that is maintained for at least about 6 months to about 12 months, or about 90 days to about 24 months.

4. 4. The pharmaceutical composition of any one of claims 1 to 3, comprising about 500 cells / μl to about 10,000 cells / μl.

5. 5. The pharmaceutical composition of claim 1, wherein at least 95% of the cells co-express pre-melanosome protein (PMEL17) and cellular retinaldehyde-binding protein (CRALBP).

6. The pharmaceutical composition according to any one of claims 1 to 5, wherein the RPE cells are cells produced by ex vivo differentiation of human embryonic stem cells.

7. The pharmaceutical composition according to any one of claims 1 to 6, which is for administration by transplanting RPE cells.

8. 8. The pharmaceutical composition of claim 7, wherein transplanting RPE cells comprises injecting the RPE cells at least one papillary diameter away from the edge of the geographic atrophy (GA) lesion.

9. Transplantation of RPE cells Covering the GA lesion, covering the fovea, covering part or all of the transformation zone bordering the GA lesion, or covering the surrounding healthy tissue adjacent to the GA lesion 9. The pharmaceutical composition of claim 1, comprising injecting RPE cells into one or more of the following:

10. RPE cells (a) culturing human embryonic stem cells or induced pluripotent stem cells in a medium containing nicotinamide to generate differentiated cells; (b) culturing the differentiated cells in a medium containing nicotinamide and activin A to generate cells that further differentiate into the RPE lineage; and (c) culturing the cells, which further differentiate into the RPE lineage, in a medium containing nicotinamide but not activin A. are cells produced by A pharmaceutical composition according to any one of claims 1 to 9.

11. 11. The pharmaceutical composition of claim 10, wherein the embryonic stem cells or induced pluripotent stem cells are grown in a medium containing bFGF and TGFβ under non-adherent conditions.

12. The pharmaceutical composition of claim 10, wherein the medium of (a) is substantially free of activin A.

13. The pharmaceutical composition according to any one of claims 1 to 12, characterized in that the cells are used to be administered in a single dose.

14. The pharmaceutical composition according to any one of claims 1 to 13, characterized in that the cells are administered to the subretinal space of a subject.

15. The pharmaceutical composition according to any one of claims 1 to 14, which is for administration by cannula.

16. 16. The pharmaceutical composition of any one of claims 1 to 15, used in combination with an immunosuppressant, wherein the immunosuppressant is administered to the subject 1 day to 3 months after administration of the RPE cells.

17. 17. The pharmaceutical composition of any one of claims 1 to 16, wherein the retinal disease or condition is selected from the group consisting of moderate dry AMD, retinitis pigmentosa, retinal detachment, retinal dysplasia, retinal atrophy, retinopathy, macular dystrophy, cone dystrophy, cone-rod dystrophy, Malattia Leventinese, Doyne honeycomb dystrophy, Sorsby's dystrophy, pattern / sphenoid dystrophy, Best vitelliform dystrophy, North Carolina dystrophy, central areolar choroidal dystrophy, angioid streaks, toxic maculopathy, Stargardt disease, pathologic myopia, retinitis pigmentosa, and macular degeneration.

18. A pharmaceutical composition for treating or slowing the progression of a retinal disease or disorder, comprising approximately 50,000 to 500,000 RPE cells as an active substance, the composition being formulated for transplantation into an area overlying a geographic atrophy (GA) lesion or an area overlying the fovea in a subject.

19. A pharmaceutical composition for stabilizing the RPE in a subject with a retinal disease or disorder, comprising approximately 50,000 to 500,000 RPE cells as an active substance, the composition being formulated for transplantation into an area overlying a geographic atrophy (GA) lesion or an area overlying the fovea in the subject.

Images