Methods and compositions for treating retinal diseases and conditions - Patents.com

Abstract

Provided herein are methods, compositions, and devices for treating ocular diseases and illnesses, including retinal conditions such as macular degeneration.

Description

[Technical field]

[0001] Related Applications This application claims the benefit of priority under 35 U.S.C. §119(e) to U.S. Provisional Patent Application No. 63 / 208,921, filed June 9, 2021, and U.S. Provisional Patent Application No. 63 / 350,175, filed June 8, 2022, the entire contents of each of which are incorporated herein by reference in their 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] background Dysfunction, degeneration and loss of RPE cells are hallmarks of retinal diseases such as AMD, Stargardt's disease, Best's disease and subtypes of retinitis pigmentosa (RP). AMD is the leading cause of visual impairment in the Western world. Among people over 75 years of age, 25-30% suffer from age-related macular degeneration (AMD), with progressive central visual field loss leading to blindness in 6-8% of patients. AMD involves multiple etiological risk factors such as aging, smoke and complement polymorphisms, and its pathophysiological root causes can be summarized as RPE aging, oxidative stress, parainflammation, Bruch's membrane aging and choroidal ischemia, which individually or collectively cause metabolic deterioration of retinal health. Retinal degeneration primarily involves the macula, the central part of the retina responsible for fine visual details, color perception, face recognition, reading and driving. There are two forms of advanced AMD: wet or exudative AMD and atrophic AMD. Dry AMD or intermediate AMD is the most prevalent form of AMD, accounting for approximately 85-90% of cases. Wet AMD is the less common of the two progressive forms, accounting for approximately 10-15% of cases. The dry form of AMD is initiated by the dysfunction of the RPE and the formation of drusen deposits beneath or above the RPE or within Bruch's membrane, which consists of metabolic end products. The disease gradually progresses to an advanced stage of geographic atrophy (GA), which involves degeneration and eventual cell death of both RPE and photoreceptor cells, causing central vision loss. In addition, the degenerated RPE affects the blood-retinal barrier (BRB), which is composed of an inner and outer barrier. The outer BRB refers to the barrier formed at the retinal pigment epithelial cell layer along with Bruch's membrane, which regulates solutes and nutrients from the choroid into the subretinal space. The outer BRB plays an essential role in maintaining the anatomical and functional integrity of the photoreceptor cells, especially within the macular region, where the highest oxygen metabolic activity in the body is carried out. The primary goal of hRPE cell therapy is to replace lost or damaged host RPE and deliver functional, active and viable RPE to support photoreceptors.

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

[0005] Dry age-related macular degeneration (AMD) progressing to atrophic AMD or geographic atrophy is the leading cause of blindness in adults in developed countries. Nearly all cases of wet AMD begin as dry AMD. Dry AMD typically affects both eyes. Currently, there are no U.S. Food and Drug Administration (FDA) or European Medicines Agency (EMA) approved treatment options available for patients with dry AMD. Preventative measures include vitamin / mineral supplements. These reduce the risk of developing wet AMD but do not affect the onset of geographic atrophy progression. Wet AMD is a repair response to avoid atrophic AMD. Unfortunately, this is an exaggerated response in the majority of cases, leading to exudation, inflammation and scarring, followed by vision loss in a relatively short time. Appropriate treatment with anti-vascular endothelial growth factor (VEGF) drugs can control the exudation and its associated harm, prevent scarring, and allow these eyes to take their natural course of gradually atrophying. Thus, most eyes with wet AMD that are successfully treated will eventually develop atrophic AMD. Summary of the Invention

[0006] overview FIELD OF THE DISCLOSURE Embodiments herein relate generally to methods, compositions, and devices for treating ocular diseases and illnesses, including retinal conditions such as macular degeneration.

[0007] In some aspects, the disclosure provides a method for assessing progression of an area of ​​retinal atrophy in a subject's retina following transplantation of retinal pigment epithelial (RPE) cells, comprising: a) defining an area of ​​geographic atrophy, or complete RPE and outer retinal atrophy (cRORA), within the retinal external limiting membrane (ELM) boundary at a first time point; b) marking and measuring the ELM boundary or ELM boundary descent using optical coherence tomography (OCT), where the ELM boundary is the boundary of atrophy and the ELM boundary descent is a definition of an area of ​​near complete histological photoreceptor depletion; c) calculating the area contained within the ELM boundary to define a first calculated area; and d) determining a square root transformation (SQRT) of the first calculated area; and defining the progression of atrophy by comparing the SQRT of the first calculated area to a control.

[0008] In some embodiments, the method further comprises repeating steps a) through c) at a second time point to determine the SQRT of a second calculated area, the control being the SQRT of the second calculated area.

[0009] In some embodiments, the control is the past progression of the retina. In some embodiments, the control is the progression of a control retina. In some embodiments, the control retina is an untreated retina of the subject.

[0010] In some embodiments, the measurement of the ELM boundary and the calculation of the first calculated area are performed manually. In some embodiments, the measurement of the ELM boundary is performed automatically by a standalone algorithm by the OCT device. In some embodiments, the measurement and calculation of the ELM boundary are performed using artificial intelligence for automatic detection, detection of areas and volumes by specific layers, and prediction of growth.

[0011] In some embodiments, the atrophy is incomplete RPE and outer retinal atrophy (iRORA) according to the Atrophy Conference Classification (CAM) Study Group Consensus Classification.

[0012] In some embodiments, the progression of the area of ​​retinal atrophy is measured in mm 2 and SQRT.

[0013] In some embodiments, steps a) through c) are performed at a third time point.

[0014] In some embodiments, the first, second and third time points are about 12 months, about 24 months and about 36 months, respectively, after implantation.

[0015] In some embodiments, the past progression is the predicted growth according to the past data of the atrophic area, and a SQRT linear growth calculation is used to predict the theoretical size of the atrophic area at any future time point.

[0016] In some embodiments, the control group to which the rate of growth of geographic atrophy is compared is a theoretical prediction of growth in the same eye.

[0017] In some embodiments, the comparison of atrophy area is in mm 2 and SQRT are performed between the treated eye and the fellow eye of the subject. In some embodiments, the calculation is 2 It is carried out in units.

[0018] In some embodiments, the comparison of areas of atrophy is performed for multiple eyes.

[0019] In some embodiments, the first time point is before transplantation of RPE cells. In some embodiments, the first time point is at the time of transplantation of RPE cells. In some embodiments, the first time point is after transplantation of RPE cells.

[0020] In other aspects, the disclosure provides methods for assessing retinal recovery or regeneration in areas within an atrophic region, comprising: a) defining and using OCT biomarkers as boundaries of any retinal layer; b) using OCT to mark and measure boundaries of any retinal layer; c) calculating the length / width and volume of specific retinal layers; d) defining the level of recovery or regeneration by comparing ELM areas calculated from steps (a)-(c); and e) detecting newly present ELM areas.

[0021] In some embodiments, the retinal layer is an ONL and the method detects a newly present ONL region. In some embodiments, the retinal layer is an OPL and the method detects a newly present OPL region. In some embodiments, the retinal layer is an ellipsoid zone and the method detects a newly present ellipsoid zone region. In some embodiments, the retinal layer is a photoreceptor cell and the method detects a newly present photoreceptor cell region.

[0022] In some embodiments, the retinal layer is an RPE cell layer and the method detects newly existing RPE regions.

[0023] In some embodiments, the retinal layers are calculated in combination.

[0024] In some embodiments, OCT examinations are performed at about 12 months, about 24 months, and about 36 months.

[0025] In some embodiments, the comparison of retinal layers is performed on the same eye. In some embodiments, the comparison of the area of ​​atrophy is performed between the treated eye and the fellow eye. In some embodiments, the comparison of the area of ​​atrophy is performed between the treated eye and the control eye.

[0026] In some embodiments, the comparison of areas of atrophy is performed for multiple eyes.

[0027] In some embodiments, the area of ​​RPE recovery is where the ELM, ONL and OPL are all present.

[0028] In yet another aspect, the disclosure provides a method for assessing clinical improvement, wherein the clinical improvement is selected from the group consisting of BCVA normal light and low light with or without computer assistance, microperimetry, reading speed, color test with or without computer assistance, flicker test, cone sensitivity and rod sensitivity.

[0029] In some embodiments, the area of ​​retinal atrophy is advanced geographic atrophy, early geographic atrophy, high-risk AMD, or late intermediate AMD.

[0030] In one aspect, the disclosure provides a method of treating or slowing the progression of a retinal disease or disorder, comprising administering a cellular therapeutic agent to a subject in need thereof, wherein the cellular therapeutic agent comprises retinal pigment epithelial (RPE) cells, and wherein the RPE cells restore the anatomy or functionality of the subject's retina.

[0031] In some embodiments, the RPE cells are derived from pluripotent cells. In some embodiments, the RPE cells are human RPE cells. In some embodiments, the RPE cells are derived from human embryonic stem cell (hESC) cell lines. In some embodiments, the RPE cells are derived from human induced pluripotent stem cells (hiPSCs) from the patient itself, other donor patients, or HLA banks of iPSCs.

[0032] In some embodiments, the RPE cells are obtained by enriching the RPE population in hypoxic (5%) culture supplemented with high concentrations of activin A, transforming growth factor beta (TGF-b) family members and nicotinamide, followed by switching to normal oxygen (20%) culture.

[0033] In some embodiments, RPE cells secrete PEDF at a concentration of about 2000 ng / ml / day to about 4000 ng / ml / day.

[0034] In some embodiments, the cellular therapeutic agent is administered to the patient in an area of ​​atrophic retina or adjacent to an area of ​​atrophic retina.

[0035] In some embodiments, the cellular therapy is administered at a dose of about 50,000 cells to about 1,000,000 cells. In some embodiments, the cellular therapy is administered at a dose of about 100,000 cells to about 750,000 cells. In some embodiments, the cellular therapy is administered at a dose of about 200,000 cells to about 500,000 cells.

[0036] In some embodiments, administration of the cellular therapeutic agent reduces the area of ​​atrophy in the subject's atrophic retina.

[0037] In some embodiments, administration of a cellular therapeutic agent restores one or more retinal layers of the retina.

[0038] In some embodiments, administration of a cellular therapeutic agent restores functionality of photoreceptor cells in the retina.

[0039] In some embodiments, administration of a cellular therapeutic agent restores the outer nuclear layer (ONL) of the retina.

[0040] In some embodiments, administration of a cellular therapeutic agent restores the ellipsoid zone (EZ) of the retina.

[0041] In some embodiments, administration of the cellular therapeutic agent restores the fovea of ​​the retina.

[0042] In some embodiments, administration of a cellular therapeutic agent restores the blood-retinal barrier (BRB) of the retina.

[0043] In some embodiments, administration of a cellular therapeutic agent remodels the extracellular matrix (ECM) of the retina.

[0044] In some embodiments, restoration of retinal anatomy or functionality is determined by assessing one or more of: reduced growth of geographic atrophy, improved visual acuity, improved reading speed, improved retinal structure, reduction in drusen, or stable engraftment of cells.

[0045] In some embodiments, the improvement is measured by microperimetry.

[0046] In some embodiments, the subject's visual acuity is improved by treatment, and the improved visual acuity is assessed by one or more of: change in total area of ​​GA lesions; change in monocular reading speed; change in Functional Reading Independence Index (FRII) composite score; change in normal luminance best-corrected visual acuity score (NL-BCVA); change in low luminance best-corrected visual acuity score (LL-BCVA); change in low luminance deficiency (LLD); change in monocular limiting print size; change in National Eye Institute Visual Functioning Questionnaire 25 item version (NEIVFQ-25) distance activity subscale score; change in number of scotomas; change in macular sensitivity; change in color vision testing, and change in systemic plasma concentrations of APL-2.

[0047] In some embodiments, the methods result in minimal or no delayed inflammation of rejection of transplanted cells.

[0048] In some embodiments, administering comprises delivering RPE cells to an area of ​​the retina or adjacent to the retina, hi some embodiments, delivering comprises transplanting RPE cells to an area of ​​the retina or adjacent to the retina.

[0049] In some embodiments, treating comprises a pluripotent secretory effect of RPE cells.

[0050] In some embodiments, the subject is afflicted with a retinal disease condition selected from dry or atrophic AMD, retinitis pigmentosa, Usher syndrome, vitelliform maculopathy, Stargardt disease, retinal detachment, retinal dysplasia, retinal atrophy, retinopathy, macular dystrophy, cone dystrophy, cone-rod dystrophy, Malattia Leventinese, Doyne honeycomb dystrophy, Sorsby dystrophy, pattern / sbutterfly dystrophy, Best vitelliform dystrophy, North Carolina dystrophy, central areolar choroidal dystrophy, angioid streaks, angiomatous striata, toxic maculopathy, pathologic myopia, and macular degeneration.

[0051] In some embodiments, the cellular therapeutic agent is administered using a delivery device.

[0052] In some embodiments, the cellular therapeutic agent is administered at or adjacent to geographic atrophy of the retina using a delivery device.

[0053] In some embodiments, the delivery device comprises a needle, a capillary and a tip. In some embodiments, the delivery device comprises a needle having an outer diameter of about 0.63 mm and an inner diameter of about 0.53 mm, a capillary having an outer diameter of about 0.5 mm and an inner diameter of about 0.25 mm, and a tip having an outer diameter of about 0.12 mm and an inner diameter of about 0.07 mm.

[0054] In another aspect, the present disclosure provides a delivery device for use in any of the methods described herein.

[0055] In some embodiments, the delivery device comprises a needle, a capillary and a tip.

[0056] In some embodiments, the device comprises a needle having an outer diameter of about 0.63 mm and an inner diameter of about 0.53 mm, a capillary having an outer diameter of about 0.5 mm and an inner diameter of about 0.25 mm, and a tip having an outer diameter of about 0.12 mm and an inner diameter of about 0.07 mm.

[0057] In yet another aspect, the present disclosure provides a composition comprising a cellular therapeutic agent for restoring retinal anatomy or functionality in a subject according to the present disclosure.

[0058] In some embodiments, new areas of RPE are defined by a change in the IR image as new areas of greyish depigmentation.

[0059] In some embodiments, the present disclosure provides a method for assessing the progression of areas of retinal atrophy in the retina following transplantation of retinal pigment epithelial (RPE) cells, comprising the steps of: defining areas of geographic atrophy within the retinal external limiting membrane (ELM) border or complete RPE and outer retinal atrophy (cRORA), defining the ELM border as the border of atrophy using optical coherence tomography (OCT), where the ELM border is accepted as the delimitation of an area with near-complete histological photoreceptor depletion; marking and measuring the ELM border or ELM descent with OCT at each examination; and determining the area (e.g., mm2) contained within the ELM border at each examination. 2 The method may include calculating the area of ​​atrophy (units) and utilizing a square root transformation (SQRT) of the calculated area to assess change over time relative to the eye itself or relative to a control eye; and defining the progression of atrophy by comparing the calculated area of ​​atrophy between two or more different tests using the OCT and ELM boundaries as boundaries.

[0060] In some embodiments, the area of ​​atrophy may be defined by a border or area of ​​preserved ONL.

[0061] In some embodiments, the area of ​​atrophy may be defined by a border or area of ​​preserved OPL.

[0062] In some embodiments, the area of ​​atrophy may be defined by a border or area of ​​preserved RPE.

[0063] In some embodiments, the area of ​​atrophy may be defined by a combination of any or all of the preserved borders or areas of the ONL, ELM, OPL and RPE.

[0064] In some embodiments, the method may involve manually measuring and calculating the ELM boundary and the area within the boundary.

[0065] In some embodiments, measurement and calculation of the ELM boundary is performed automatically by the OCT device, by a standalone algorithm, and optionally using artificial intelligence for automatic detection by specific layers, area and volume detection, and prediction of growth.

[0066] In some embodiments, the atrophy can be incomplete RPE and outer retinal atrophy (iRORA) according to the Atrophy Conference Classification (CAM) Study Group Consensus Classification.

[0067] In some embodiments, the change in atrophy area is measured in mm 2 and SQRT.

[0068] In some embodiments, testing is performed at about 12 months, about 18 months, about 24 months, and about 36 months, respectively.

[0069] In some embodiments, in some aspects, the comparison of the atrophic area uses predicted growth from historical data of the atrophic area and uses a SQRT linear growth calculation to predict the theoretical size of the atrophic area at any future time point.

[0070] In some embodiments, the control group to which the rate of growth of geographic atrophy is compared is a theoretical prediction of growth in the same eye.

[0071] In some embodiments, the comparison of atrophy area is in mm 2 and SQRT may be performed between the treatment and fellow eyes.

[0072] In some embodiments, the comparison of atrophy area is in mm 2 and SQRT may be performed between treated and control eyes.

[0073] In some embodiments, the comparison of areas of atrophy is performed on multiple eyes.

[0074] In some embodiments, the first time point can be before transplantation of RPE cells. In some embodiments, the first time point can be at the time of transplantation of RPE cells. In some embodiments, the first time point can be after transplantation of RPE cells.

[0075] In some embodiments, the time before transplantation can vary to encompass multiple time points ranging from one to days, weeks, or years. In some embodiments, the time after transplantation can vary to encompass multiple time points ranging from one to days, weeks, or years.

[0076] In some embodiments, the second time point is after the first time point. Thus, the second time point can be anywhere from 1 week to 10 years after the first time point. In some embodiments, the second time point can be anywhere from 1 week to 10 years after transplantation of RPE cells.

[0077] In some embodiments, the present disclosure provides a method for assessing retinal recovery or regeneration in areas within atrophic regions, using OCT and, optionally, one or more stand-alone algorithms using OCT and optionally artificial intelligence for automated detection of areas and volumes by specific layers and prediction of growth or kinetics. Assessment of recovery or regeneration may be performed by one or more examinations of the retina, the method comprising the steps of defining and using OCT biomarkers as boundaries of any retinal layer; marking and measuring the boundaries of any retinal layer using OCT; calculating the length / width and volume of the specific retinal layer; defining the level of recovery or regeneration by comparing the ELM areas calculated from steps (a)-(c); and detecting newly existing ELM areas.

[0078] In some embodiments, the retinal layer is an ONL and the method detects a newly present ONL region. In some embodiments, the retinal layer is an OPL and the method detects a newly present OPL region. In some embodiments, the retinal layer is an ellipsoid zone and the method detects a newly present ellipsoid zone region. In some embodiments, the retinal layer is a photoreceptor cell and the method detects a newly present photoreceptor cell region. In some embodiments, the retinal layer is an RPE cell layer and the method detects a newly present RPE region.

[0079] In some embodiments, the retinal layers are calculated in combination.

[0080] In some embodiments, OCT examinations are performed at about 12 months, about 18 months, about 24 months, and about 36 months.

[0081] In some embodiments, the comparison of retinal layers is performed on the same eye. In some embodiments, the comparison of areas of atrophy is performed between the treated eye and the fellow eye. In some embodiments, the comparison of areas of atrophy is performed between the treated eye and the control eye. In some embodiments, the comparison of areas of atrophy is performed on multiple eyes.

[0082] In some embodiments, the area of ​​RPE recovery includes when the ELM, ONL and OPL are all present.

[0083] In some embodiments, the disclosure provides a method for determining areas of geographic atrophy or complete RPE and outer retinal atrophy (cRORA) within the retinal external limiting membrane (ELM) border; determining the ELM border as the border of atrophy using optical coherence tomography (OCT), where ELM border descent or ELM border is accepted as the delimitation of an area with near-complete histological photoreceptor depletion; marking and measuring the ELM border or ELM descent with OCT at each examination; determining the area (e.g., mm2 and utilizing a square root transformation (SQRT) of the calculated area to assess change over time relative to the eye itself or relative to a control eye; and defining the progression of atrophy by comparing the calculated atrophy area between two or more different exams using the OCT and ELM boundaries as boundaries.

[0084] In some embodiments, the present disclosure provides methods for assessing clinical improvement by defining and using OCT biomarkers as boundaries of any retinal layer; using OCT to mark and measure boundaries of any retinal layer; calculating the length / width and volume of specific retinal layers; defining the level of recovery or regeneration by comparing the ELM areas calculated from steps (a)-(c); and detecting newly existing ELM areas.

[0085] In some embodiments, the clinical improvement is selected from BCVA normal light and low light with or without computer assistance, microperimetry, reading speed, color test with or without computer assistance, flicker test, cone sensitivity and rod sensitivity.

[0086] In some embodiments, the area of ​​retinal atrophy is advanced geographic atrophy, early geographic atrophy, high-risk AMD, or late intermediate AMD.

[0087] The retinal pigment epithelium (RPE) is a monolayer of neuroepithelial-derived pigment cells located on Bruch's membrane between photoreceptor outer segments (POS) and the choroidal vasculature. The RPE monolayer is critical for photoreceptor function and health. Dysfunction, damage, and loss of retinal pigment epithelial (RPE) cells are hallmarks of certain ocular diseases and disorders, such as age-related macular degeneration (AMD), inherited macular degenerations including Stargardt disease, Best disease (early-onset vitelliform macular dystrophy), and subtypes of retinitis pigmentosa (RP). Transplantation of RPE into the retina of individuals affected by such diseases can be used as a cell replacement therapy in retinal diseases in which the RPE has degenerated.

[0088] Human pluripotent stem cells offer significant advantages as a source of RPE cells for transplantation. Their pluripotent developmental potential allows their differentiation into true functional RPE cells, and considering the possibility of infinite self-renewal, they may serve as an infinite source of RPE cells. Indeed, it has been demonstrated that human embryonic stem cells (hESCs) and human induced pluripotent stem cells (iPSCs) can differentiate into RPE cells in vitro, attenuate retinal degeneration, and maintain visual function after subretinal transplantation. Thus, hESCs may be an unlimited source for the production of RPE cells for cell therapy.

[0089] However, most cell-based treatments are usually cryopreserved in low-temperature solutions that are not compatible with direct administration into the body, creating practical problems for clinical use. Cells should be transplanted within a few hours after thawing, or they may begin to lose viability and quality. Furthermore, cells must be prepared prior to administration in a certified facility that may not be in close proximity to a clinical site, hospital or other treatment facility. Finally, preparation of the final formulation is considered part of the cell therapy manufacturing process, so each subject's treatment dose must be released by a qualified technician.

[0090] The present disclosure addresses these and other shortcomings in the fields of regenerative medicine and RPE cell therapy. The present disclosure further provides data regarding various methods, devices and compositions.

[0091] Teachings, methods, compositions, devices, and know-how for embodiments of the present invention can be found in International Publication No. WO 2019 / 130061, entitled "RETINAL PIGMENT EPITHELIUM CELL COMPOSITIONS," published on July 4, 2019; International Publication No. WO 2018 / 170494, entitled "METHODS FOR MEASURING THERAPEUTIC EFFECTS OF RETINAL DISEASE THERAPIES," published on September 20, 2018; and International Publication No. WO 2017 / 017686, entitled "LARGE SCALE PRODUCTION OF RETINAL PIGMENT EPITHELIAL CELLS," published on February 2, 2017, each of which is incorporated herein by reference in its entirety for all of its methods, devices, and compositions, alone or in combination with each other. [Brief description of the drawings]

[0092] [Figure 1] Figure 1 shows a retinal scan showing areas of pigmentation (arrows) within the geographic atrophy (GA) of subject 18 3 months after treatment with RPE cells, demonstrating the presence of RPE cells in the inferior region of the GA. The area of ​​RPE cell transplantation, represented by the open circle, is also known as the bleb area, a blister-like formation resulting from the injection of RPE cells.

[0093] [Diagram 2] Figure 2 shows a retinal scan showing areas of pigmentation (arrows) within the GA of subject 18 9 months after treatment, demonstrating the presence of RPE cells in the inferior region of the GA.

[0094] [Diagram 3] 1 is a graph showing the change in visual acuity based on the change in Early Treatment of Diabetic Retinopathy Study (ETDRS) letters from baseline for each of the 12 subjects after the indicated treatments. Nearly all subjects maintained their baseline BCVA, and more than half had a steady improvement in BCVA.

[0095] [Figure 4] 4 is a graph showing the mean change in size of GA (mm2) from baseline over time for treated and untreated eyes (fellow eyes) of cohort 4. The data demonstrate that GA growth was slower in treated eyes compared to fellow eyes.

[0096] [Diagram 5] Figure 5 is a graph showing the change in visual acuity based on the mean change in ETDRS letters from baseline over time for treated and untreated (fellow) eyes in Cohort 4. The data demonstrate that BCVA loss was less severe in the treated eye compared to the fellow eye.

[0097] [Figure 6] 6 is a graph showing the mean change from baseline in ETDRS letter count over time for the treated and untreated eyes (fellow eyes) of subject 22. The subject showed substantial improvement and gain in visual function activity in the treated eye versus decline in the other eye.

[0098] [Figure 7] 7A-7C show the changes over time for subject 14. FIG. 7A is a graph showing the mean change in ETDRS letter count from baseline over time for the treated and untreated eyes (fellow eyes). FIG. 7B is a graph showing the mean change in GA (mm2) size from baseline over time for the treated and untreated eyes (fellow eyes). FIG. 7C shows the number of letters read at baseline and 3 years after treatment in the treated and untreated eyes (fellow eyes). The subject showed substantial differences in both anatomical and visual functional aspects between the treated and fellow eyes in favor of the treated eye.

[0099] [Figure 8] 8 is a graph showing the change in reading speed (words per minute) from baseline over time in the treated eye (left panel) and untreated eye (fellow eye, right panel) of an individual subject from Cohort 4. The data demonstrate functional clinical vision improvement in the treated eye versus the fellow eye.

[0100] [Figure 9] Figure 9 shows high-resolution optical coherence tomography (OCT) images from the treated retina of subject 14 at baseline (top) and 9 months after treatment (bottom). The left image shows the area of ​​the retina shown in the right image. The GA border demonstrates recovery / regeneration of the outer retina at 9 months.

[0101] [Figure 10] Figure 10 shows OCT images of the treated retina of subject 14 before the start of the study (historical, orange, left panel), at baseline (red), 9 months after treatment (blue), and 23 months after treatment (yellow). Regression of GA from baseline was observed at both 9 and 23 months after treatment, demonstrating anatomical improvement and regeneration / recovery of the outer retina.

[0102] [Figure 11] FIG. 11 is a graph showing the change in total size of GA (total area in square root transformed, SQRT) for both eyes of subject 14 and the percent change in mm SQRT / year (projected growth from historical plots) from the previous year and from baseline. The yellow hatched bar shows the predicted / anticipated growth for the fellow eye (FE), which is the untreated eye. The blue hatched bar shows the predicted / anticipated growth for the study treated eye.

[0103] [Figure 12] FIG. 12 is an OCT retinal image from the treated eye of subject 14 showing the GA boundary based on the ELM boundary at baseline (top) and 3 months after treatment (bottom). The ELM boundary is indicated by the red arrow and dotted line. The change in the ELM boundary from baseline (BSL) to 3 months (3M) is indicated by the large arrow. The outer plexiform layer is indicated by the blue arrow. New RPE cells are indicated by the small green arrow in the bottom image. The left image shows the area of ​​the retina shown in the right image. The ELM boundary and / or ONL / OPL as well as new presumable central growth of RPE are already observed 3M after treatment.

[0104] [Figure 13] FIG. 13 is an OCT retinal image from the treated eye of subject 14 showing the GA boundary based on the ELM boundary at baseline (top) and 5 months after treatment (bottom). The ELM boundary is indicated by the red arrow and dotted line. The change in the ELM boundary from baseline (BSL) to 5 months (5M) is indicated by the large arrow. The outer plexiform layer is indicated by the blue arrow. New RPE cells are indicated by the small green arrow. The left image shows the area of ​​the retina shown in the right image. The ELM boundary and / or ONL / OPL as well as new presumable RPE central growth are observed 5M after treatment.

[0105] [Figure 14] FIG. 14 is an OCT retinal image from the treated eye of subject 14 showing the GA boundary based on the ELM boundary at baseline (top), 9 months after treatment (middle) and 23 months after treatment (bottom). The ELM boundary is indicated by the red arrow and dotted line. The change in the ELM boundary from baseline (BSL) to 9 months (9M) is indicated by the large arrow. The change from 9M to 23 months (23M) is indicated by the medium arrow. The outer plexiform layer is indicated by the blue arrow. New RPE cells are indicated by the small green arrow. The left image shows the area of ​​the retina shown in the right image. The ELM boundary and / or ONL / OPL as well as central growth of new presumable RPE are observed at 9M after treatment with slight regression at 23M.

[0106] [Figure 15] FIG. 15 shows the changes in microperimetry testing of the treated eye of subject 14 at 23 months (23M) and 35 months (35M) after treatment. FIG. 15 demonstrates the improvement in visual function and reduction in scotoma ("blind spot / area" represented as a black stain on an orange circle), as well as improved light sensitivity at 35M compared to 23M. Microperimetry is a fundus-related visual field test that captures specific visual areas in the macular region, producing a high-resolution and precise mapping of the retinal sensitivity area. Microperimetry is a better test to assess changes in visual function with higher reliability than "simple" BCVA testing. Furthermore, microperimetry provides an accurate correlation between anatomical changes and visual function deficits.

[0107] [Figure 16] FIG. 16 is an OCT retinal image from the treated eye of subject 21 showing the GA border based on the ELM border at baseline (top) and 1 month after treatment (bottom). The ELM border is indicated by the arrow and dotted line. The OPL border is indicated by the arrow. The change in the ELM border from baseline (BSL) to 1 month (1M) is indicated by the arrow between the dotted lines. The left image shows the area of ​​the retina shown in the right image. Central growth of the ELM border and / or OPL is observed 1M after treatment.

[0108] [Figure 17] Figure 17 is an infrared (IR) image of the retina of subject 21. GA boundaries at baseline and 1 month are shown.

[0109] [Figure 18] FIG. 18 is an OCT retinal image from the treated eye of subject 21 showing an isolated atrophic lesion at baseline (top) and 3 months after treatment (bottom). The left image shows the area of ​​retina shown in the right image. New features (circled) suggest regeneration of the outer retina at 3 months. Near complete recovery of the previous atrophic area was observed, with regeneration of the defect layer and "disappearance" of the atrophic lesion.

[0110] [Figure 19] Figure 19 shows OCT retinal images from the treated eye of subject 21 showing GA at baseline (top) and 3 months after treatment (bottom). The left image shows the area of ​​the retina shown in the right image. The new highly reflective monolayer likely represents RPE cells, and after 3 months the ELM, OPL, and ONL may have recovered.

[0111] [Figure 20]Figure 20 is an OCT retinal image from the treated eye of subject 21 showing GA at baseline (top) and 3 months after treatment (bottom). The left image shows the area of ​​retina shown in the right image. A very thin but homogenous and continuous layer of ONL (circled), with preserved ELM and RPE monolayers over areas of choroidal hypertransparency, normally absent, was observed 3 months after treatment. This indicates a restored new layer within the atrophic area.

[0112] [Figure 21] Figure 21 shows images of isolated atrophic lesions in the retina of subject 21 before (baseline, top left), one month after (middle left), and two months after (bottom left) administration of OpRegen-RPE. The right image shows the area of ​​the retina shown in the left image.

[0113] [Figure 22] Figure 22 shows images of the superior GA region of the retina of subject 21 before (baseline, top left), one month after (middle left), and two months after (bottom left) administration of OpRegen-RPE. The right image shows the region of the retina shown in the left image.

[0114] [Figure 23] FIG. 23 shows the time course of subject 22. The left panel is a graph showing the mean change in ETDRS letter count from baseline over time for the treated and untreated eyes (fellow eyes). The right panel is a graph showing the mean change in GA (mm2) size from baseline over time for the treated and untreated eyes (fellow eyes). The data show substantial differences in both anatomical and visual function aspects between the treated and fellow eyes in favor of the treated eye. Substantial visual acuity improvement was observed in the treated eye.

[0115] [Figure 24] FIG. 24 is a fundus photography (FP) image showing fine pigment movement in the retina of subject 22 at 3 months post-treatment (right panel) but not at baseline (left panel), indicating the presence of RPE cells at 3 months.

[0116] [Diagram 25] Figure 25 is an IR image of the retina at baseline (left) and 3 months after treatment (right) of subject 22. The GA boundary is reduced and less defined at 3 months.

[0117] [Figure 26] Figure 26 is an OCT retinal image from the treated eye of subject 22 showing central GA at baseline (top) and 3 months post-treatment (bottom). The left image shows the area of ​​retina shown in the right image. The baseline border of atrophy is indicated with a line. New features are indicated with small arrows including reduced subsidence of the outer plexus, new ELM within the atrophic area, new RPE within the atrophic area, and reduced hypertransmission.

[0118] [Figure 27] Figure 27 is an OCT retinal image from the treated eye of subject 22 showing inferior GA at baseline (top) and 3 months post-treatment (bottom). The left image shows the area of ​​retina shown in the right image. The baseline border of atrophy is indicated with a line. New features are indicated with small arrows including reduced subsidence of the outer plexus, new ELM within the atrophic area, and new RPE within the atrophic area.

[0119] [Figure 28] Figure 28 is an OCT retinal image from the treated eye of subject 22 showing an isolated atrophic lesion at baseline (top) and 3 months after treatment (bottom). The left image shows the area of ​​retina shown in the right image. The baseline border of atrophy is indicated with a line. New features are indicated with small arrows including reduced subsidence of the outer plexus, new ELM within the atrophic area, and new RPE within the atrophic area.

[0120] [Figure 29] Figure 29 shows OCT retinal images showing the boundaries of GA at baseline (left) and 3 months (right) based on the ELM boundaries. Total area, growth rate, and SQRT transformed growth rate are shown.

[0121] [Diagram 30]Figure 30 is an OCT retinal image showing the central GA region of subject 22 at baseline (top left), 2 months (middle left) and 3 months (bottom left) after treatment. New subretinal material (RPE cells) was observed at 2 months, and increased subretinal material and reformation of the ELM was observed at 3 months (arrows). The right image shows the area of ​​the retina shown in the left image. The blue circle marks the same location and is a progressive coordinate showing the choroidal vessels that will be used to capture the exact area of ​​the retina at subsequent visits.

[0122] [Diagram 31] FIG. 31 shows retinal images showing the area of ​​RPE delivery in subject 14 at baseline (top left), intraoperatively (Intra OP, top right), 2 months (bottom left) and 3 months (bottom right) after treatment and prior to treatment. The bleb represents the area of ​​cell delivery. The bleb covered the GA during surgery, showing complete coverage of the GA by RPE cells.

[0123] [Diagram 32] Figure 32 are retinal images showing intraoperative images of blebs representing the area of ​​RPE cell delivery for subjects 19 (left) and 21 (right). The GA is indicated by the arrow.

[0124] [Figure 33A] 33A-33C are spectral domain optical coherence tomography (SD-OCT) images: Fig. 33A shows an exemplary B-scan. [Figure 33B] FIG. 33B is a B-scan from FIG. 33A with the boundaries between the layers superimposed. [Figure 33C] FIG. 33C is a B-scan from FIG. 33A with the boundary between the layers superimposed.

[0125] [Diagram 34] Figure 34 shows an example of thickness and area maps generated from SD-OCT. Tissue loss is indicated by white areas, while preserved tissue areas are indicated by gray or black. Relative thicknesses of the whole retina (left panel), outer nuclear layer (second from left), photoreceptor outer segment (second from right), and RPE + drusen complex (right panel) are shown.

[0126] [Diagram 35] Figure 35 shows total retinal thickness maps of treated (top) and untreated (bottom) eyes from subject 8 at baseline (left), 3 months (second from left), 6 months (second from right), and 12 months (right) after treatment. The average total thickness is shown.

[0127] [Diagram 36] Figure 36 shows thickness maps of the outer nuclear layer (ONL) of treated (top) and untreated (bottom) eyes from subject 8 at baseline (left), 3 months (second from left), 6 months (second from right), and 12 months (right) after treatment. Total area of ​​the ONL is shown.

[0128] [Figure 37] Figure 37 shows photoreceptor outer segment thickness maps of treated (top) and untreated (bottom) eyes from subject 8 at baseline (left), 3 months (second from left), 6 months (second from right), and 12 months (right) after treatment. Total photoreceptor outer segment area is shown.

[0129] [Figure 38] Figure 38 shows RPE-drusen complex thickness maps for treated (top) and untreated (bottom) eyes from subject 8 at baseline (left), 3 months (second from left), 6 months (second from right) and 12 months (right) after treatment. The total area of ​​the RPE-drusen complex is shown.

[0130] [Figure 39] Figure 39 shows total retinal thickness maps of treated (top) and untreated (bottom) eyes from subject 5 at baseline (left), 6 months (center) and 12 months (right) after treatment. The average total thickness is shown.

[0131] [Diagram 40] Figure 40 shows thickness maps of the outer nuclear layer (ONL) of treated (top) and untreated (bottom) eyes from subject 5 at baseline (left), 6 months (center) and 12 months (right) after treatment. The total area of ​​the ONL is shown.

[0132] [Diagram 41] Figure 41 shows photoreceptor outer segment thickness maps of treated (top) and untreated (bottom) eyes from subject 5 at baseline (left), 6 months (center) and 12 months (right) after treatment. Total photoreceptor outer segment area is shown.

[0133] [Diagram 42] Figure 42 shows RPE-drusen complex thickness maps for treated (top) and untreated (bottom) eyes from subject 5 at baseline (left), 6 months (center) and 12 months (right) after treatment. The total area of ​​the RPE-drusen complex is shown.

[0134] [Diagram 43] Figure 43 shows total retinal thickness maps of treated (top) and untreated (bottom) eyes from subject 13 at baseline (left), 6 months (center) and 12 months (right) after treatment. The average total thickness is shown.

[0135] [Diagram 44] Figure 44 shows thickness maps of the outer nuclear layer (ONL) of treated (top) and untreated (bottom) eyes from subject 13 at baseline (left), 6 months (center), and 12 months (right) after treatment. The total area of ​​the ONL is shown.

[0136] [Diagram 45] Figure 45 shows photoreceptor inner segment thickness maps of treated (top) and untreated (bottom) eyes from subject 13 at baseline (left), 6 months (center) and 12 months (right) after treatment. Total photoreceptor outer segment area is shown.

[0137] [Figure 46] Figure 46 shows photoreceptor outer segment thickness maps of treated (top) and untreated (bottom) eyes from subject 13 at baseline (left), 6 months (center) and 12 months (right) after treatment. Total photoreceptor outer segment area is shown.

[0138] [Figure 47]Figure 47 shows RPE-drusen complex thickness maps for treated (top) and untreated (bottom) eyes from subject 13 at baseline (left), 6 months (center) and 12 months (right) after treatment. The total area of ​​the RPE-drusen complex is shown.

[0139] [Figure 48] Figure 48 shows total retinal thickness maps of treated (top) and untreated (bottom) eyes from subject 14 at baseline (left) and 12 months after treatment (right). Mean total thickness is shown.

[0140] [Figure 49] Figure 49 shows thickness maps of the outer nuclear layer (ONL) of treated (top) and untreated (bottom) eyes from subject 14 at baseline (left) and 12 months after treatment (right). Total area of ​​the ONL is shown.

[0141] [Figure 50] Figure 50 shows photoreceptor inner segment thickness maps of treated (top) and untreated (bottom) eyes from subject 14 at baseline (left) and 12 months after treatment (right). Total photoreceptor outer segment area is shown.

[0142] [Figure 51] 51 shows photoreceptor outer segment thickness maps of treated (top) and untreated (bottom) eyes from subject 14 at baseline (left) and 12 months after treatment (right). Total photoreceptor outer segment area is shown.

[0143] [Figure 52] Figure 52 shows RPE-drusen complex thickness maps for treated (top) and untreated (bottom) eyes from subject 14 at baseline (left) and 12 months after treatment (right). The total area of ​​the RPE-drusen complex is shown.

[0144] [Diagram 53]Figure 53 shows a baseline FA examination in subject 8, in which a large amount of fluorescein dye had leaked into the vitreous cavity, thereby blocking the visibility of vascular perfusion during the choroidal flush and arterial phase, suggesting pre-existing blood-retinal barrier disruption and parainflammation in the eye. At 22 months post-implantation, the FA examination showed clear choroidal and retinal vascular perfusion with no dye leaking into the vitreous cavity, indicating that OpRegen had restored the integrity of the disrupted BRB, likely through multiple mechanisms of action.

[0145] [Figure 54A] Figures 54A-54D show four cases with similar changes or improvements in FA imaging between baseline and 10.5 months to 22 months post-implantation. [Figure 54B] See legend to Figure 54A. [Fig. 54C] See legend to Figure 54A. [Fig. 54D] See legend to Figure 54A.

[0146] [Figure 55] Figure 55 shows that drusen disappearance started in the superior graft area (upper left) and moved inferiorly to clear almost the entire posterior region, except for a small elongated band that remained 8 months postoperatively (top, second from left, large circle). OCT imaging features were consistent with color fundus photographs at 5.5 months (top, second from right) and 8 months (bottom, second from right) compared to baseline (upper right and lower right), with subRPE drusen significantly reduced or disappeared.

[0147] [Figure 56A] Figure 56A: FA showed a significant reduction in staining (drusen) but appeared to have a veil-like membrane obscuring the retinal vasculature. Pericytic reaction was seen. [Figure 56B] FIG. 56B: At 22 months on color fundus examination, the retinal tissue appears clearer compared to that at baseline. [Figure 56C]FIG. 56C shows that at 11 months, after the large drusen had disappeared, the graft continued to remodel the host retina.

[0148] [Figure 57] FIG. 57 provides a time course of FA examination from early, middle and late stages, demonstrating significant improvement in retinal health, better visibility of vascular perfusion throughout, and reduced inflammation, with the retinal tissue appearing much cleaner.

[0149] [Figure 58] FIG. 58 shows OpRegen cell therapy in GA scarring and ECM remodeling.

[0150] [Figure 59] FIG. 59 shows OCT images of different forms of ECM remodeling.

[0151] [Figure 60A] Figure 60A and Figure 60B show two tables showing visual function in subjects of cohort 4 by measuring the change in number of letters in the ETDRS test from baseline to 6M hours. Figure 60A represents visual function of the treated eye and Figure 60B represents visual function of the fellow eye. Baseline is represented by 0, and positive numbers (also marked in green) represent the number of letters gained from baseline. Negative numbers (also marked in red) are represented by a minus in front of the number and represent the number of letters lost from baseline. For example, subject 13 (602) maintained stable BCVA improvement and gained 19 letters from baseline at the last visit. [Figure 60B] See legend to Figure 60A.

[0152] [Figure 61]FIG. 61A is an image of the fundus from color photography and infrared photography. The top left image shows a color fundus photograph at baseline. The top right image shows atrophy at baseline in red and atrophy 14 months prior to baseline in orange, with natural progression from orange to red. The bottom left color fundus photograph image shows a light brownish rim around the lesion, corresponding to the location of OpRegen cells 15 months after transplantation. The bottom right infrared image shows the atrophic area 15 months after transplantation in green, with the surrounding gray rim corresponding to the area of ​​OpRegen cells and area or retinal repair. FIG. 61B is an image from optical coherence tomography. The top image shows an OCT scan across the atrophic area 15 months after transplantation, with the red area being the RPE, the dark blue area being the ELM, and the light blue area being the ONL. These layers have progressed to the center for retinal repair compared to the same scan but taken at baseline. The bottom image shows the same scan at baseline with much larger areas of atrophy not showing the RPE, ELM and ONL.

[0153] [Figure 62] Figure 62 shows the potential reduction in lesion size following OpRegen cell transplantation. Red: baseline border of the lesion. Blue: 8 months.

[0154] [Figure 63] Figure 63 shows the presence of a new highly reflective monolayer in the area of ​​former CRORA that is likely viable RPE, as it exhibits features of the ELM, ONL, and OPL.

[0155] [Figure 64] FIG. 64 shows consistent findings of retinal regeneration (new ONL, EM, ELZ, and RPE): new regions of the ONL are defined using the ELM and / or OPL borders.

[0156] [Figure 65]FIG. 65 is a graph showing the change in total size of atrophic lesions in SQRT for both eyes, and the percent change in mmSQRT / year from previous and baseline (plot of projected growth rate from historical).

[0157] [Figure 66] FIG. 66 shows a schematic diagram of OpRegen, an allogeneic suspension of RPE cells with the potential to counteract RPE cell loss in GA.

[0158] [Figure 67] FIG. 67 shows that OpRegen has the potential to combat RPE cell loss in the GA region by supporting retinal structure and function.

[0159] [Figure 68A] Figures 68A and 68B are graphs showing evidence of improvement in visual function, where in cohort 4, the mean gain was 7.6 letters and 25% of patients gained 15 letters or more. Figure 68A shows the graph for cohorts 1-3 and Figure 68B shows the graph for cohort 4. [Figure 68B] See legend to Figure 68A.

[0160] [Figure 69] FIG. 69 is an image showing subretinal delivery of OpRegen to the GA area and fovea, where greater improvements in visual function were observed with areas of improvement in outer retinal structures. Five patients in cohort 4 had OpRegen delivered to most or all of the GA area, including the fova. Five patients had greater improvements in visual function (mean gain of 12.8 letters) with evidence of clearly improved areas of outer retinal structures as assessed by SD-OCT.

[0161] [Figure 70]Figure 70 shows images of evaluation of GA after OpRegen delivery, demonstrating the advantage of SD-OCT versus fundus autofluorescence (FAF) imaging. Allogeneic hESC-derived RPE cells in OpRegen were younger and had lower lipofuscin content. It was expected that OpRegen RPE cells would not be easily detectable by standard FAF after subretinal delivery.

[0162] [Figure 71] Figure 71 shows images of greater hyperreflectivity visible in the RPE / Bruch's membrane. SD-OCT images suggested the presence of OpRegen in areas of previous GA.

[0163] [Figure 72] Figure 72 shows an example image of the improvement of outer retinal structure by SD-OCT when OpRegen is delivered to the GA region. The focal disruption of the RPE layer, choroidal hyperpermeability and outer subretinal retention at baseline were no longer present at 12 months. The enrollment scan was confirmed by the presence of prominent drusen and choroidal vascular markings.

[0164] [Figure 73] Figures 73A and 73B are images showing an example of improvement in outer retinal structure by SD-OCT when OpRegen is delivered to the GA region. Figure 73A is an image showing cRORA resolution near the baseline GA border. Figure 73B is an image showing a comparison at 12 months compared to baseline and the following observations: cRORA features were no longer present. There was greater hyperreflectivity at the RPE / Bruch's membrane level, reduced choroidal hypertransmission, greater continuity of the outer retina, and resolution of subretinal pooling. Similar features were seen at the nasal, superior, and inferior borders of the GA.

[0165] [Figure 74] FIG. 74 shows an example of improvement in outer retinal structure by SD-OCT when OpRegen is delivered to the GA region.

[0166] [Figure 75] Figure 75 is an image showing the results for subject number 120 at baseline. The BCVA in the treated eye (OS) was 54, the same as baseline and 4 letters more than the previous year (50). The BCVA on the fellow eye was 28, a 33 letter decrease from baseline (61) and an increase of 7 letters from the previous year (21). The repair layer was still preserved and cells were still present. FAF showed rapid proliferation in the fellow eye when compared to the treated eye.

[0167] [Figure 76] Figure 76 is an image showing the results at the 4-year visit for subject #120. BCVA in the treated eye (OS) was 54, the same as baseline and 4 letters more than the previous year (50). BCVA on the fellow eye was 28, a 33 letter decrease from baseline (61) and an increase of 7 letters from the previous year (21). The repair layer was still preserved and cells were still present. FAF showed rapid proliferation in the fellow eye when compared to the treated eye.

[0168] [Figure 77] Figure 77 is an image showing the results at the 4-year visit for subject #120. BCVA in the treated eye (OS) was 54, the same as baseline and 4 letters more than the previous year (50). BCVA on the fellow eye was 28, a 33 letter decrease from baseline (61) and an increase of 7 letters from the previous year (21). The repair layer was still preserved and cells were still present. FAF showed rapid proliferation in the fellow eye when compared to the treated eye.

[0169] [Figure 78] Figure 78 is an image showing the results of the fellow eye. The BCVA in the treated eye (OS) was 54, the same as baseline and 4 letters more than the previous year (50). The BCVA on the fellow eye was 28, a 33 letter decrease from baseline (61) and an increase of 7 letters from the previous year (21). The repair layer was still preserved and cells were still present. FAF showed rapid proliferation in the fellow eye when compared to the treated eye.

[0170] [Figure 79] Figure 79 shows images showing the results at baseline and 9-month visits. The BCVA in the treated eye (OS) was 54, the same as baseline and 4 letters more than the previous year (50). The BCVA on the fellow eye was 28, a 33 letter decrease from baseline (61) and an increase of 7 letters from the previous year (21). The repair layer was still preserved and cells were still present. FAF showed rapid proliferation in the fellow eye when compared to the treated eye.

[0171] [Figure 80] Figure 80 shows images showing the results at baseline and 4-year visits. The BCVA in the treated eye (OS) was 54, the same as baseline and 4 letters more than the previous year (50). The BCVA on the fellow eye was 28, a 33 letter decrease from baseline (61) and an increase of 7 letters from the previous year (21). The repair layer was still preserved and cells were still present. FAF showed rapid proliferation in the fellow eye when compared to the treated eye.

[0172] The figures provide various illustrations and examples of surprising and unexpected results. The embodiments relate to various methods, which may include any of the assessments and assays discussed, described, or for which data are presented in the figures. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0173] Detailed Description FIELD OF THE DISCLOSURE Embodiments of the specification relate generally to methods, compositions, and devices for treating ocular diseases and ailments, including retinal conditions such as macular degeneration.

[0174] In some embodiments, the compositions, methods and devices may utilize allogeneic ("off the shelf") product candidates. For example, this may mean that the material is derived from cell lines rather than individual patients, facilitating large scale production and lower production costs than patient-specific treatments.

[0175] The methods, apparatus, compositions, etc. may include those described in the accompanying drawings.

[0176] After reading this description, it will be clear to a person skilled in the art how to implement the present disclosure in various alternative embodiments and alternative applications. However, not all of the various embodiments of the present invention are described herein. It is understood that the embodiments presented herein are presented by way of example only and are not limiting. Therefore, this detailed description of various alternative embodiments should not be interpreted as limiting the scope or breadth of the present disclosure described herein.

[0177] Before disclosing and describing the present technology, it is to be understood that the embodiments described below are not limited to particular compositions, methods of preparing such compositions, or uses thereof, and as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0178] The detailed description is divided into various sections solely for the convenience of the reader, and disclosures found in any section may be combined with those of another section. Titles or subtitles may be used herein for the convenience of the reader and are not intended to affect the scope of the disclosure.

[0179] definition The term "treating" or "treatment" refers to any indication of success in treating or ameliorating an injury, disease, pathology, or condition, including any objective or subjective parameter, such as alleviation, mitigation, attenuation of symptoms, or making the injury, pathology, or condition more tolerable to the patient, slowing the rate of decline or decay, reducing the decline at the end point of decline, improving the patient's physical or mental health, etc. Treatment or amelioration of a condition shall be based on objective or subjective parameters, including the results of a physical exam, a neuropsychiatric exam, and / or a psychiatric evaluation. The term "treating" and conjugations thereof may include prevention of an injury, pathology, condition, or disease. In embodiments, treating is preventing. In embodiments, treating does not include preventing. As used herein, "treating" or "treatment" also broadly includes any approach to obtaining beneficial or desired results in a subject's condition, including clinical results (as is well understood in the art). Beneficial or desired clinical results include, but are not limited to, alleviation or amelioration of one or more symptoms or conditions, reduction in the extent of disease, stabilization of the disease state (i.e., not worsening), prevention of disease infection or spread, delay or slowing of disease progression, amelioration or alleviation of symptoms, reduction of disease recurrence, and remission, whether partial or total, detectable or undetectable. In other words, "treatment" as used herein includes cure, amelioration, or prevention of disease. Treatment may include preventing the onset of disease, preventing the spread of disease, alleviating the symptoms of disease, completely or partially eliminating the underlying cause of disease, shortening the duration of disease, or a combination thereof.

[0180] As used herein, "treating" and "treatment" include prophylactic treatment. The treatment method includes administering a therapeutically effective amount of an active agent to the subject. The administration step may consist of a single administration or may include a series of administrations. The length of the treatment period will depend on various factors such as the severity of the condition, the age of the patient, the concentration of the active agent, the activity of the composition used for treatment, or a combination thereof. It will also be understood that the effective amount of the agent used for treatment or prevention may increase or decrease over the course of a particular treatment or prevention regimen. Changes in dosage may occur and be evident by standard diagnostic assays known in the art. In some embodiments, chronic administration may be required. For example, the composition of the present invention is administered to the subject in an amount and for a period sufficient to treat the patient. In some embodiments, the treating or treatment is not a prophylactic treatment.

[0181] The term "prevent" refers to a reduction in the occurrence of disease symptoms in a patient. As noted above, prevention may be complete (no detectable symptoms) or may be partial, such that fewer symptoms are observed than would occur without treatment.

[0182] "Patient" or "subject in need thereof" means an organism suffering from or susceptible to a disease or condition that can be treated by administration of a pharmaceutical composition as provided herein. Non-limiting examples include humans, other mammals, cows, rats, mice, dogs, monkeys, goats, sheep, cattle, deer, and other non-mammals. In some embodiments, the patient is a human.

[0183] An "effective amount" is an amount sufficient for the composition to achieve a stated purpose compared to the absence of the composition (e.g., achieve the effect for which it is administered, treat a disease, reduce enzyme activity, increase enzyme activity, reduce a signal transduction pathway, or reduce one or more symptoms of a disease or condition). An example of an "effective amount" is an amount sufficient to contribute to the treatment, prevention, or alleviation of one or more symptoms of a disease, also referred to as a "therapeutically effective amount". "Alleviation" of one or more symptoms (and grammatical equivalents of this phrase) means a reduction in the severity or frequency of one or more symptoms, or the elimination of one or more symptoms. A "prophylactically effective amount" of a drug (e.g., a cell described herein) is an amount of drug that, when administered to a subject, has an intended prophylactic effect, such as preventing or delaying the onset (or recurrence) of an injury, disease, pathology, or condition, or reducing the likelihood of the onset (or recurrence) of an injury, disease, pathology, or condition, or a symptom thereof. A complete prophylactic effect does not necessarily occur with the administration of one dose, but may occur only after the administration of a series of doses. Thus, a prophylactically effective amount can be administered in one or more doses. As used herein, "activity-reducing amount" refers to the amount of antagonist required to reduce the activity of an enzyme compared to the absence of antagonist. As used herein, "function-disrupting amount" refers to the amount of antagonist required to destroy the function of an enzyme or protein compared to the absence of antagonist. The exact amount varies depending on the purpose of treatment and can be ascertained by those skilled in the art using known techniques (see, for example, Lieberman, Pharmaceutical Dosage Forms (vols.1-3, 1992); Lloyd, The Art, Science and Technology of Pharmaceutical Compounding (1999); Pickar, Dosage Calculations (1999); and Remington: The Science and Practice of Pharmacy, 20th Edition, 2003, Gennaro, Ed., Lippincott, Williams & Wilkins).

[0184] For any composition described herein, the therapeutically effective amount may be initially determined from cell culture assays. The target concentration is the concentration of active composition (e.g., cell concentration or cell number) that can achieve the methods described herein, as measured using methods described herein or known in the art.

[0185] As is well known in the art, the therapeutically effective amount for human use can also be determined from animal models.For example, the dosage for human can be formulated to achieve the concentration that has been found to be effective in animals.The dosage in human can be adjusted by monitoring the effectiveness of the composition and adjusting the dosage upward or downward as described above.It is well within the capabilities of a person skilled in the art to adjust the dosage to achieve maximum effectiveness in human based on the above and other methods.

[0186] The term "therapeutically effective amount" as used herein refers to an amount of a therapeutic agent sufficient to improve the above-mentioned disorder. For example, for a given parameter, a therapeutically effective amount shows at least a 5%, 10%, 15%, 20%, 25%, 40%, 50%, 60%, 75%, 80%, 90% or at least a 100% increase or decrease. The therapeutic effect can also be expressed as a "-fold" increase or decrease. For example, a therapeutically effective amount can have at least a 1.2-fold, 1.5-fold, 2-fold, 5-fold or more effect over the control.

[0187] Dosage may vary depending on the requirements of the patient and the composition employed. In the context of this disclosure, the dosage administered to the patient shall be sufficient to provide a beneficial therapeutic response to the patient over an extended period of time. The size of the dosage will also be determined by the existence, nature and extent of any adverse side effects. Determining the appropriate dosage for a particular situation is within the skill of the physician. Generally, treatment is initiated with smaller dosages that are less than the optimal dosage of the composition. This dosage is then increased by small increments until the optimal effect is obtained according to the situation. Dosage and intervals can be individually adjusted to provide a level of the administered composition that is effective for the particular clinical indication being treated. This provides a treatment plan that is commensurate with the severity of the individual's condition.

[0188] "Co-administration" means that the compositions described herein are administered simultaneously with, immediately before, or immediately after the administration of one or more additional therapies. The compositions provided herein can be administered alone or co-administered to a patient. Co-administration means that the compositions are administered individually or in combination (multiple compositions) simultaneously or sequentially. Thus, the preparations can also be combined with other active substances (e.g., to reduce metabolic degradation) if necessary.

[0189] "Control" or "control experiment" is used according to its plain ordinary meaning and refers to an experiment in which the experimental subjects or reagents are treated as in a parallel experiment, except for the omission of an experimental procedure, reagent, or variable. In some cases, a control is used as a standard of comparison in evaluating experimental effects. In some embodiments, a control is a measure of protein activity in the absence of a composition described herein (including embodiments and examples).

[0190] "Pharmaceutically acceptable excipient" and "pharmaceutically acceptable carrier" refer to substances that aid in the administration and absorption of active agents to and by a subject and may be included in the compositions of the present disclosure without causing significant adverse toxic effects to the patient. Non-limiting examples of pharmaceutically acceptable additives include water, NaCl, normal saline, lactated Ringer's solution, normal sucrose, normal glucose, binders, fillers, disintegrants, lubricants, coatings, sweeteners, flavorings, saline (such as Ringer's solution), alcohol, oils, gelatin; carbohydrates such as lactose, amylose, starch, fatty acid esters, hydroxymethylcellulose, polyvinylpyrrolidine, dyes, and the like. Such preparations may be sterilized and mixed with auxiliary agents such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for affecting osmotic pressure, buffers, coloring agents, and / or aromatic substances that do not adversely react with the compositions of the present disclosure, as appropriate. Those skilled in the art will recognize that other pharmaceutical additives are useful in the present disclosure.

[0191] "Cell" as used herein refers to a cell that performs metabolic or other functions sufficient to preserve or replicate its genomic DNA. Cells can be identified by methods well known in the art, including, for example, the presence of an intact membrane, staining with a particular dye, the ability to produce progeny, or in the case of gametes, the ability to combine with a second gamete to produce viable progeny. Cells can include prokaryotic and eukaryotic cells. Prokaryotic cells include, but are not limited to, bacteria. Eukaryotic cells include, but are not limited to, yeast cells and cells from plants and animals, such as mammalian, insect (e.g., spodoptera) and human cells. Cells can be useful when they are naturally non-adherent or have been treated to prevent them from adhering to surfaces, for example, by trypsinization.

[0192] As used herein, "stem cells" refer to cells that can remain in an undifferentiated state (e.g., pluripotent or multipotent stem cells) in culture for extended periods of time until induced to differentiate into other cell types (e.g., fully differentiated cells) with specific specialized functions. In some embodiments, "stem cells" include embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs), adult stem cells, mesenchymal stem cells, and hematopoietic stem cells. In some embodiments, RPE cells are generated from pluripotent stem cells (e.g., ESCs or iPSCs).

[0193] As used herein, "induced pluripotent stem cells" or "iPSCs" refer to stem cells that have been genetically engineered into somatic cells to express, for example, genes encoding 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. In addition, iPSCs can be generated using non-integrative methods, for example, by using small molecules or RNA.

[0194] 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 phrase "embryonic stem cells" includes cells obtained from embryonic tissues (e.g., blastocysts) formed after conception before implantation of the embryo (i.e., pre-implantation blastocysts), expanded blastocyst cells (EBCs) obtained from blastocysts at post-implantation / pre-gastrulation stage (see WO 2006 / 040763), and embryonic germ (EG) cells obtained from fetal reproductive tissues at any time during conception, preferably before 10 weeks of conception. In some embodiments, embryonic stem cells are obtained using well-known cell culture methods. For example, human embryonic stem cells can be isolated from human blastocysts.

[0195] It is understood that commercially available stem cells may also be used in aspects and embodiments of the present disclosure. Human ES cells may 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, CY1O, 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, HUES56, HUES57, HUES58, HUES59, HUES60, HUES61, HUES62, HUES63, HUES64, HUES65, HUES66, HUES67, 25, HUES26, HUES27, HUES28, CyT49, RUES3, WAO1, UCSF4, NYUES1, NYUES2, NYUES3, NYUES4, NYUESS, NYUES6, NYUES7, UCLA1, UCLA2, UCLA3, WA077(H7), WA09(H9), WA13(H13), WA14(H14), HUES62, HUES63, HUES64, CT1, CT2, CT3, CT4, MA135, Eneavour-2, WIBR1, WIBR2, WIBR3, WIBR4, WIBRS, WIBR6, HUES45, Shef3, Shef6, BINhem19, BJNhem20, SAGO1, SAOO1.

[0196] The term "retinal pigment epithelium" or "RPE", also known as the "pigmented layer of the retina", refers to the pigmented layer of cells on the outer retina. The RPE layer is located between Bruch's membrane (the inner choroid) and the photoreceptor cells. The RPE is an intermediate for providing nutrients to the retina and aids in many functions including retinal development, light absorption, secretion of growth factors, and mediating the immune response of the eye. Dysfunction of the RPE can result in vision loss or blindness in conditions including retinitis pigmentosa, diabetic retinopathy, West Nile virus, and macular degeneration.

[0197] The term "disease" or "condition" refers to a state or health condition of a patient or subject that can be treated with the compositions or methods provided herein. Age-related macular degeneration, or AMD, is a progressive, chronic disease of the central retina and is the leading cause of vision loss worldwide. Most vision loss occurs in the later stages of the disease due to one of two processes: neovascularization ("wet" AMD) and geographic atrophy (GA, "dry"). In GA, progressive atrophy of the retinal pigment epithelium, choriocapillaris, and photoreceptor cells occurs. While the wet form of AMD is more common (85-90% of all cases), it can progress to the "wet" form, which, if left untreated, results in rapid and severe vision loss. 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 Contributing factors include both environmental and genetic factors. The pathogenesis of the disease involves abnormalities in four functionally interrelated tissues: the retinal pigment epithelium (RPE), Bruch's membrane, the choriocapillaris, and photoreceptor cells. However, impaired RPE cell function is an early and key event in the molecular pathway that leads to clinically relevant AMD changes. Currently, there is no approved treatment for dry AMD. Preventive measures include vitamin / mineral supplements. These reduce the risk of developing wet AMD but do not affect the onset of geographic atrophy (GA) progression.

[0198] A non-limiting list of diseases for which the effect of treatment may be measured according to the methods provided herein 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 dystrophies and other dystrophies of the RPE, Stargardt's disease, RPE and retinal damage due to injury caused by any one of actinic, laser, inflammatory, infectious, radiation, neovascular or traumatic causes, retinal dysplasia, retinal atrophy, retinopathy, macular dystrophies, cone dystrophies, cone-rod dystrophies, honeycomb retinal dystrophies (Malattia nephrectomy), and other conditions that may affect the retina. Leventinese), Doyne honeycomb dystrophy, Sorsby dystrophy, pattern / butterfly dystrophy, Best disease, North Carolina dystrophy, central ring choroidal dystrophy, angioid streaks, toxic maculopathy, pathologic myopia, retinitis pigmentosa, and macular degeneration. In some embodiments, the disease is dry AMD. In some embodiments, the disease is GA.

[0199] "Geographic atrophy" or "GA" or "atrophic retina", also known as dry age-related macular degeneration (AMD) or advanced dry AMD, is an advanced form of age-related macular degeneration that can result in progressive and irreversible loss of the retina (photoreceptors, retinal pigment epithelium, choriocapillaris) that can result in loss of visual function over time.

[0200] In some embodiments, the RPE deficiency may be due to one or more of: advanced age, smoking, unhealthy weight, low antioxidant intake, or cardiovascular disorder. In other embodiments, the RPE deficiency may be due to a congenital abnormality. "Retinal pigment epithelial cells," "RPE cells," and "RPE," which may be used interchangeably as the context permits, refer to cells of a cell type that resembles, for example, functionally, epigenetically, or by expression profile, the native RPE cells that form the pigment epithelial cell layer of the retina (e.g., upon implantation, administration, or delivery into the eye, they exhibit functional activity similar to that of native RPE cells).

[0201] As used herein, the term "OpRegen" refers to a lineage-restricted human RPE cell line. RPE cells are induced under differentiation medium supplemented with activin A, transforming growth factor beta (TGF-b) family and nicotinamide to enrich for RPE populations. OpRegen is a single cell suspension formulated in either ophthalmic balanced salt solution (BSS Plus) or ready-to-administer (RTA) thawed injection (TAI) formulation, for example, as described in PCT Publication No. WO 2019 / 130061, which is incorporated by reference in its entirety for all formulations, compositions, methods, reagents, etc.

[0202] Treatment FIELD OF THE DISCLOSURE Embodiments of the specification relate generally to methods, compositions, and devices for treating ocular diseases and ailments, including retinal conditions such as macular degeneration.

[0203] Thus, in one aspect, there is provided a method of treating or slowing the progression of a retinal disease or disorder as described, illustrated or exemplified herein.

[0204] According to some embodiments, treating retinal disease or slowing its progression can be demonstrated by microperimetry to evaluate vision regeneration. Microperimetry is one of the tools that can be used to measure or evaluate visual function with high resolution mapping of visual sensitivity area. Microperimetry allows to identify this specific visual area or the location of visual impairment on the retina, and can "bridge the gap" between anatomical and clinical changes, with good correlation between these two important parameters (anatomical defects and visual impairment).

[0205] According to other embodiments, restoring microperimetrically assessed vision includes demonstrating that administration of RPE cells results in improved microperimetric assessment compared to a baseline microperimetric assessment. According to other embodiments, restoring microperimetrically assessed vision includes demonstrating that administration of RPE cells results in preserved microperimetric assessment compared to baseline and the fellow / untreated eye.

[0206] According to certain embodiments, treating or slowing the progression of retinal disease comprises a reduction in GA lesion growth rate of about 5% to about 20% one year after administration of the RPE cells compared to baseline or the fellow eye. In some embodiments, treating or slowing the progression of retinal disease comprises a reduction in GA lesion growth rate of about 5% to about 50% one year after administration compared to baseline or the fellow eye. In some embodiments, treating or slowing the progression of retinal disease comprises a reduction in GA lesion growth rate of about 5% to about 25% one year after administration compared to baseline or the fellow eye. In some embodiments, treating or slowing the progression of retinal disease comprises a reduction in GA lesion growth rate of about 5% to about 100% one year after administration compared to baseline or the fellow eye. In some embodiments, treating or slowing the progression of retinal disease comprises a reduction in GA lesion growth rate of about 5% to about 10% one year after administration compared to baseline or the fellow eye. The amount may be any value or subrange within the recited range, including the endpoint.

[0207] According to some embodiments, treating or slowing the progression of retinal disease includes one or more of stable best corrected visual acuity (BCVA); no decline in low luminance test performance; or no decline in microperimetry sensitivity; or no decline in readout speed. In some embodiments, the comparison is with an age-matched and gender-matched control. In some embodiments, the comparison is with a baseline. In some embodiments, the comparison is with a fellow eye. In some embodiments, the comparison is performed over a period of about 1 week to about 5 years. In some embodiments, the comparison is performed at about 1 month. In some embodiments, the comparison is performed at about 3 months. In some embodiments, the comparison is performed at about 6 months. In some embodiments, the comparison is performed at about 1 year. The period may be any value or subrange within the recited range, including the endpoint.

[0208] According to some embodiments, a pharmaceutical composition for treating or slowing the progression of a retinal disease or disorder is provided, comprising about 25,000 to about 1,000,000 RPE cells as an active substance. In some embodiments, the composition comprises about 50,000 to about 500,000 RPE cells. In some embodiments, the composition comprises about 100,000 to about 500,000 RPE cells. In some embodiments, the composition comprises about 250,000 to about 500,000 RPE cells. In some embodiments, the composition comprises about 50,000 to about 400,000 RPE cells. In some embodiments, the composition comprises about 50,000 to about 300,000 RPE cells. In some embodiments, the composition comprises about 50,000 to about 250,000 RPE cells. In some embodiments, the composition comprises about 50,000 to about 200,000 RPE cells. The amount may be any value or subrange within the recited range, including the endpoints.

[0209] In some embodiments, the methods include administering a cellular therapeutic agent to a subject in need of restoration of retinal structure in a retinal disease, wherein the cellular therapeutic agent is capable of restoring retinal structure in a retinal disease.

[0210] Cell Therapy In some aspects, the present disclosure is a cell therapy comprising retinal pigment epithelial (RPE) cells derived from pluripotent cells. Such cell therapy includes, but is not intended to be limited to, OpRegen.

[0211] According to some embodiments, the RPE cells express at least one, two, three, four or five markers of mature RPE cells. According to some embodiments, the 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, the RPE cells may also express a marker of RPE progenitor cells (e.g., MITF). In other embodiments, the RPE cells express PAX-6. In other embodiments, the RPE cells express at least one marker of retinal progenitor cells, including, but not limited to, Rx, OTX2 or SIX3. Optionally, the RPE cells may express SIX6 and / or LHX2.

[0212] According to some embodiments, the RPE cells are OpRegen® cells.

[0213] 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.

[0214] 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.

[0215] According to other embodiments, the RPE cells have a morphology similar to that of native 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.

[0216] According to some embodiments, the RPE cells are generated from pluripotent stem cells (eg, ESCs or iPSCs).

[0217] Induced pluripotent stem cells (iPSCs) have been developed by genetic manipulation of somatic cells, for example, by expressing genes encoding 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. In addition, iPSCs can be generated using non-integrative methods, for example, by using small molecules or RNA.

[0218] Human embryonic stem cells may be isolated from human blastocysts. Human blastocysts are typically obtained from human in vivo preimplantation embryos or in vitro fertilized (IVF) embryos. Alternatively, single-cell human embryos may be expanded to the blastocyst stage. To isolate 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 cultured in tissue culture flasks containing an appropriate medium that allows its proliferation. After 9-15 days, the ICM-derived outgrowths are dissociated into clumps by either mechanical dissociation or enzymatic degradation, and the cells are then reseeded in fresh tissue culture medium. Colonies that exhibit undifferentiated morphology are individually selected by micropipette, mechanically dissociated into clumps, and reseeded. The resulting ES cells are then routinely split every 4-7 days. For further 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].

[0219] Furthermore, ES cells have been used to infect mice (Mills and Bradley, 2001), golden hamsters [Doetschman et al., 1988, Dev Biol. 127:224-7], rats [Iannaccone et al., 1994, Dev Biol. 163:288-92], rabbits [Giles et al. 1993, Mol Reprod Dev. 36:130-8; Graves and Moreadith, 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 Suppl. Dev. 6:563-8; Mitalipova et al., 2001, Cloning. 3:59-67] and from other species, such as non-human primate species (rhesus monkeys and marmosets) [Thomson et al., 1995, Proc Natl Acad Sci US A. 92:7844-8; Thomson et al., 1996, Biol Reprod. 55:254-9].

[0220] Expanded blastocyst cells (EBCs) can be obtained from blastocysts at least 9 days post-fertilization, at the pre-gastrulation stage. Prior to culturing the blastocysts, the zona pellucida is digested (e.g., Tyrode's acid solution method (Sigma Aldrich, St Louis, MO, USA)) to expose the inner cell mass. The blastocysts are then cultured as whole embryos in vitro for at least 9 days post-fertilization and up to 14 days (i.e., prior to the gastrulation event) using standard embryonic stem cell culture methods.

[0221] Another method for preparing ES cells is described in Chung et al., Cell Stem Cell, Volume 2, Issue 2, 113-117 (February 7, 2008). This method involves removing a single cell from an embryo during the in vitro fertilization process. The embryo is not destroyed in the process.

[0222] EG (embryonic germ) cells are prepared from primordial germ cells obtained from fetuses at approximately 8-11 weeks of gestation (for 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 cell morphology consistent with EG cells is observed (typically after 7-30 days or 1-4 passages). For further details regarding methods for preparing human EG cells, see Shamblott et al., [Proc. Natl. Acad. Sci. USA 95:13726, 1998] and U.S. Patent No. 6,090,622.

[0223] Yet another method for preparing ES cells is by parthenogenesis, a process that also does not destroy the embryo.

[0224] ES culture methods may include the use of a feeder cell layer that secretes factors necessary for stem cell proliferation while inhibiting their differentiation. Culturing is typically performed on a solid surface, such as a surface 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 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 medium to maintain ESCs in an undifferentiated state. Such growth factors include bFGF and / or TGFs. In other embodiments, agents can be added to the medium to maintain hESCs in a naive, undifferentiated state - see, e.g., Kalkan et al., 2014, Phil. Trans. R. Soc. B, 369:20130540.

[0225] Human umbilical cord fibroblasts may be grown in Dulbecco's Modified Eagle 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 may be done using methods known in the art (e.g., Gamma cell, 220 Exel, MDS Nordion 3,500-7500rads). Once sufficient cells are obtained, they may be frozen (e.g., cryopreserved). For the expansion of ESCs, human umbilical cord fibroblasts are typically grown in DMEM (e.g., SH30081.01, Hyclone) supplemented with about 20% human serum (and glutamine) at a concentration of about 25,000-100,000 cells / cm2 on gelatin (e.g., recombinant human gelatin (RhG 100-001, fibrin) or human vitronectin or laminin 521 (Bio hESCs are seeded on a solid surface (e.g., T75 or T175 flasks) optionally coated with an adhesive substrate such as lamina. hESCs are typically plated on top of feeder cells after 1-4 days in supportive medium (e.g., NUTRISTEM® or NUT(+) with human serum albumin). Additional factors may be added to the medium to prevent differentiation of ESCs, such as bFGF and TGFI3. Once a sufficient amount of hESCs is obtained, the cells can be mechanically disrupted (e.g., by using a sterile tip or a disposable sterile stem cell tool; 14602 Swemed). Alternatively, the 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, the hESCs are removed using TrypLE Select, and after the second round of expansion, the hESCs are removed using collagenase A.

[0226] The ESCs may be grown on feeders prior to the differentiation step. Non-limiting examples of feeder layer-based media are described herein above. The growth is typically carried out for at least 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days or 10 days. The 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, the growth is carried out for at least 2 passages to at least 20 passages. In other embodiments, the growth is carried out for at least 2 passages to at least 40 passages. After growth, the pluripotent stem cells (e.g., ESCs) are subjected to directed differentiation using a differentiation agent.

[0227] Feeder cell-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 matrix (e.g., MATRIGELR™, laminin or vitronectin) in the presence of culture media, e.g., Lonza L7 system, mTeSR, StemPro, XFKSR, E8, NUTRISTEM®). Unlike feeder-based cultures, which require simultaneous growth of feeder cells and stem cells and can result in a mixed cell population, stem cells grown in feeder-free systems are easily separated from the surface. Culture media used to grow stem cells include factors that effectively inhibit differentiation and promote their growth, such as MEF-conditioned medium, bFGF, etc.

[0228] In some embodiments, after expansion, the pluripotent ESCs are subjected to directed differentiation on an adherent surface (without intermediate generation of spheroids or enviroid bodies). See, e.g., International Patent Application Publication No. WO 2017 / 072763, which is incorporated herein by reference in its entirety.

[0229] Thus, according to one embodiment 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 directed differentiation on the adherent surface are undifferentiated ESCs and express markers of pluripotency, e.g., 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 pluripotency markers such as NANOG, Rex-1, alkaline phosphatase, Sox2, TDGF-beta, SSEA-3, SSEA-4 and / or TRA-1-81.

[0230] In one exemplary differentiation protocol, undifferentiated embryonic stem cells are differentiated into RPE cell lineages on an adherent 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, and homologous ligands including activin (e.g., activin A, activin B, and activin AB), nodal, anti-Mullerian hormone (AMH), several bone morphogenetic proteins (BMPs), e.g., BMP2, BMP3, BMP4, BMP5, BMP6 and BMP7, and growth and 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).

[0231] 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.

[0232] NA, also known as "niacinamide," is an amide derivative form of vitamin B3 (niacin) that is believed to preserve 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 in the treatment and prevention of diabetes.

[0233] According to some embodiments, the nicotinamide is a nicotinamide derivative or a nicotinamide mimetic. As used herein, the term "derivative of nicotinamide (NA)" refers to a compound that is a chemically modified derivative of natural NA. In one embodiment, the chemical modification can be a substitution of the pyridine ring of the basic NA structure (through the carbon or nitrogen member of the ring) through the nitrogen or oxygen atom of the amide moiety. When substituted, one or more hydrogen atoms may be replaced with a substituent and / or the substituent may be bonded to the N atom to form a quadrivalent positively charged nitrogen. Thus, the nicotinamide of the present invention includes substituted or unsubstituted nicotinamide. In other embodiments, the chemical modification can be the deletion or substitution of a single group, for example to form a thiobenzamide analog of NA, all as would be understood by one 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 also described in relation to the inhibitory activity of PDE4 enzyme (WO 03 / 068233; WO 02 / 060875; GB2327675A) or as VEGF receptor tyrosine kinase inhibitor (WO 01 / 55114).For example, the method of preparing 4-aryl-nicotinamide derivatives (WO 05 / 014549).Other exemplary nicotinamide derivatives are disclosed in WO 01 / 55114 and EP2128244.

[0234] Nicotinamide mimetics include modified forms of nicotinamide and chemical analogues of nicotinamide that reproduce the effect of nicotinamide in 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 are inhibitors 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.

[0235] Further contemplated differentiation agents include, for example, noggin, Wnt antagonists (Dkkl or IWR1e), nodal antagonists (Lefty-A), retinoic acid, taurine, GSK3b inhibitors (CHIR99021) and notch inhibitors (DAPT).

[0236] According to certain embodiments, differentiation is performed by: (a) culturing ESCs in a medium containing a first differentiation agent (e.g., nicotinamide); (b) culturing the cells obtained from step a) in a medium containing a member of the TGFB superfamily (e.g., activin A) and the first differentiation agent (e.g., nicotinamide).

[0237] Step (a) may be carried out in the absence of a member of the TGFI3 superfamily (eg, activin A).

[0238] In some embodiments, the medium in step (a) is completely devoid of a member of the TGFI3 superfamily. In other embodiments, the level of a member of the TGFI3 superfamily in the medium is less than 20 ng / ml, 10 ng / ml, 1 ng / ml or even less than 0.1 ng / ml.

[0239] 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 TGFI3 superfamily (e.g., activin A). This step is referred to herein as step (b*).

[0240] The above protocol will be described in further detail with additional embodiments. Step (a): Once a sufficient amount of ESCs is obtained, the differentiation process is started. The cells can be removed from the cell culture (e.g., by using collagenase A, dispase, TrypLE select, EDTA) and seeded on a non-adhesive substrate (e.g., cell culture plates such as Hydrocell or agarose-coated culture dishes, or Petri bacteriological dishes) in the presence of nicotinamide (and in 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 seeded on a non-adhesive substrate (e.g., cell culture plate), the cell culture can be referred to as a cell suspension, preferably 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., culture plate, carrier). Sources of suspension stem cells have been previously described in WO 06 / 070370, which is incorporated herein by reference in its entirety. This step may be carried out for a minimum of 1 day, more preferably 2 days, 3 days, 1 week, or even 14 days. Preferably, the cells are cultured in suspension for no more than 3 weeks with, for example, 0.01-100 mM, 0.1-100 mM, 0.1-50 mM, 5-50 mM, 5-20 mM, e.g., 10 mM nicotinamide (and in the absence of activin A). In one embodiment, the cells are cultured in suspension for 6-8 days with, for example, 0.01-100 mM, 0.1-100 mM, 0.1-50 mM, 5-50 mM, 5-20 mM, e.g., 10 mM nicotinamide (and in the absence of activin A).

[0241] According to some embodiments, when cells are cultured on a non-adherent substrate, such as a cell culture plate, the atmospheric oxygen conditions are 20%. However, manipulation of the atmospheric oxygen conditions is also contemplated, such that the atmospheric oxygen percentage 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 first cultured on a non-adherent substrate under normal atmospheric oxygen conditions, and then reduced to less than normal atmospheric oxygen conditions.

[0242] Examples of non-adherent cell culture plates include those manufactured by Nunc (eg, Hydrocell catalog number 174912).

[0243] Typically, the clusters contain at least about 50-500,000, 50-100,000, 50-50,000, 50-10,000, 50-5000, or 50-1000 cells. According to one embodiment, the cells in the cluster are not organized into layers and form irregular shapes. In one embodiment, the clusters are substantially devoid of pluripotent embryonic stem cells. In other embodiments, the clusters contain a small amount of pluripotent embryonic stem cells (e.g., 5% or less or 3% or less (e.g., 0.01-2.7%) of cells that co-express OCT4 and TRA-1-60 at the protein level). Typically, the clusters contain cells that have been partially differentiated under the influence of nicotinamide. Such cells predominantly express neural and retinal progenitor markers such as PAX6, Rax, Six3 and / or CHX10.

[0244] The clusters can be dissociated using enzymatic or non-enzymatic (e.g., mechanical) methods known in the art. According to some embodiments, the cells are dissociated such that they are no longer in clusters (e.g., aggregates or clumps of 2-100,000 cells, 2-50,000 cells, 2-10,000 cells, 2-5000 cells, 2-1000 cells, 2-500 cells, 2-100 cells, 2-50 cells). According to certain embodiments, the cells are in a single cell suspension.

[0245] The cells (e.g., dissociated cells) may then be seeded onto an adhesive substrate and cultured in the presence of nicotinamide (and in the absence of activin A), for example, at 0.01-100 mM, 0.1-100 mM, 0.1-50 mM, 5-50 mM, 5-20 mM, and for example, 10 mM. The concentrations may be any value or subrange within the recited ranges, including the endpoints. This step may be carried out for a minimum of 1 day, more preferably 2 days, 3 days, 1 week, or even 14 days. Preferably, the cells are not cultured in the presence of nicotinamide (and in the absence of activin A) for more than 3 weeks. In an exemplary embodiment, this step is carried out for 6-7 days.

[0246] According to other embodiments, when cells are cultured on an adherent substrate, such as laminin, the atmospheric oxygen conditions are 20%. They may be manipulated such that the atmospheric oxygen percentage is less than about 20%, 15%, 10%, more preferably less than about 9%, less than about 8%, less than about 7%, less than about 6%, more preferably about 5% (e.g., 1%-20%, 1%-10%, or 0-5%). The amount may be any value or subrange within the recited range, including the endpoints.

[0247] According to some embodiments, the cells are first cultured on the adherent substrate under normal atmospheric oxygen conditions, after which the oxygen is reduced to below normal atmospheric oxygen conditions.

[0248] Examples of adhesive substrates or mixtures of substances may include, but are not limited to, fibronectin, laminin, poly D-lysine, collagen and gelatin.

[0249] Step (b): After the first stage of directed differentiation (step a; i.e., culture 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), the partially differentiated cells are then cultured in the presence of activin A (e.g., 0.01-1000 ng / ml, 0.1-200 ng / ml, 1-200 ng / ml-e.g., 140 ng / ml, 15 The cells may be subjected to a further differentiation step on an adhesive substrate by culturing in the presence of 0.0 ng / ml, 160 ng / ml or 180 ng / ml of Activin A. Thus, Activin A may 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. The concentration may be any value or subrange within the recited range, including the endpoint.

[0250] Nicotinamide may also be added at this stage (e.g., 0.01-100 mM, 0.1-100 mM, 0.1-50 mM, 5-50 mM, 5-20 mM, e.g., 10 mM). The concentration may be any value or subrange within the recited range, including the endpoint. This stage may be performed for 1 day to 10 weeks, 3 days to 10 weeks, 1 week to 10 weeks, 1 week to 8 weeks, 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, at least 10 weeks. The duration may be any value or subrange within the recited range, including the endpoint.

[0251] According to some embodiments, this stage is carried out for about 8 days to about 2 weeks. This differentiation stage may be carried out under low or normal atmospheric oxygen conditions, as detailed herein above.

[0252] Step (b*): After the second stage of directed differentiation (i.e., culture in the presence of nicotinamide and activin A on an adherent substrate; step (b), the further differentiated cells are optionally subjected to a subsequent differentiation stage in adherent substrate culture in the absence of activin A and 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). The concentrations may be any value or subrange within the recited ranges, including the end points. This stage 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. This differentiation stage may also be carried out in low or normal atmospheric oxygen conditions, as detailed herein above.

[0253] The basal medium in which ESCs are differentiated can be any known cell culture medium known in the art for supporting cell growth in vitro, typically a medium that includes a defined base solution containing salts, sugars, amino acids and any other nutrients necessary to maintain cells in a viable state in 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 according to the present invention include NUTRISTEM® (without bFGF and TGF for ESC differentiation, with bFGF and TGF for ESC proliferation), NEUROBASAL™, KO-DMEM, DMEM, DMEM / F12, CELLGRO™ Stem Cell Growth Medium or X-VIVO™. The basal medium can be supplemented with various agents known in the art for dealing with cell culture. The following are non-limiting references to various supplements that may be included in the cultures used in accordance with the present disclosure: serum or serum replacement-containing media, 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 then be used to carry one or more members of the TGFI3 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, NT4.

[0254] According to some embodiments, the medium used to differentiate ESCs is NUTRISTEM® medium (Biological Industries, 06-5102-01-1A).

[0255] 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 US Patent Application No. 20130196369, the contents of which are incorporated herein by reference in their entirety.

[0256] Preparations comprising RPE cells may be prepared in accordance with Good Manufacturing Practices (GMP) (e.g., the preparations are GMP compliant) and / or Current Good Transplant Practices (GTP) (e.g., the preparations may be GTP compliant).

[0257] During the differentiation process, embryonic stem cells can be monitored for their differentiation state. Cell differentiation can be determined upon examination of cell or tissue specific markers known to indicate differentiation.

[0258] 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 immunoassay for secreted molecular markers.

[0259] After the differentiation step 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., International Patent Application Publication No. W02017 / 021973, which is 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%, at least 70% of the cells removed from the culture (and subsequently expanded) are non-pigmented cells. In other embodiments, this is done mechanically, for example, using a cell scraper. In yet other embodiments, this is done chemically (e.g., by EDTA). A combination of enzymatic and chemical treatments is 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.

[0260] According to one embodiment of the present disclosure, at least 50%, 60%, 70%, 80%, 90%, 95%, 100% of the total cells in the culture are removed and then expanded.

[0261] The growth of the mixed population of cells can be performed on extracellular matrices such as gelatin, collagen I, collagen IV, laminin (e.g., laminin 521), fibronectin and poly-D-lysine. For growth, the cells can be cultured in serum-free KOM, serum-containing medium (e.g., DMEM with 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 so that a population of purified RPE cells is obtained. Such cells show the characteristic polygonal morphology and pigmentation of RPE cells.

[0262] In one embodiment, 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. The concentrations can be any value or subrange within the recited ranges, including the endpoints.

[0263] The mixed population of cells may be grown in suspension (with or without microcarriers) or in monolayers. The growth of the mixed population of cells in monolayer or suspension cultures may be adapted for large-scale growth in bioreactors or multi / hyperstacks by methods well known to those skilled in the art.

[0264] According to some embodiments, the growth phase is carried out for at least 1-20 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 growth phase is carried out for 1 week to 10 weeks, more preferably 2 weeks to 10 weeks, more preferably 3 weeks to 10 weeks, more preferably 4 weeks to 10 weeks, or 4 weeks to 8 weeks. The duration may be any value or subrange within the recited ranges, including the endpoints.

[0265] According to yet other embodiments, the mixed population of cells is passaged at least once during a growth phase, at least twice during a growth phase, at least three times during a growth phase, at least four times during a growth phase, at least five times during a growth phase, or at least six times during a growth phase.

[0266] If the cells are enzymatically harvested, they can be allowed to continue to grow for more than 8, more than 9, or even more than 10 passages (e.g., 11-15 passages). The total number of cell doublings can be increased to more than 30, e.g., 31, 32, 33, 34 or more. (See WO 2017 / 021973, incorporated herein by reference in its entirety).

[0267] The population of RPE cells generated according to the methods described herein can be characterized according to a number of different parameters. Thus, for example, the RPE cells obtained can be polygonal in shape and pigmented.

[0268] It will be understood that the cell population and cell composition disclosed herein generally lack undifferentiated human embryonic stem cells. According to some embodiments, less than 1:250,000 cells are Oct4+TRA-1-60+ cells, for example, as measured by FACS. The cells can also downregulate (more than 5,000-fold) the expression of GDF3 or TDGF, as measured by PCR. The RPE cells of this aspect 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-beta, SSEA-3, SSEA-4, TRA-1-60 and / or TRA-1-81.

[0269] The therapeutic RPE cell preparation may be substantially purified and contain at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% RPE cells with respect to non-RPE cells. The RPE cell preparation may be essentially free of non-RPE cells or may consist of RPE cells. For example, a substantially purified preparation of RPE cells may 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 may be at about 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%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.08%, 0.07%, 0.06%, 0.05%, 0.04%, 0.03%, 0.02%, 0.05 ... 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.

[0270] RPE cell preparation can be substantially pure with respect to both non-RPE cells and other levels of maturity of RPE cells.Preparation can be substantially purified with respect to non-RPE cells and enriched with respect to mature RPE cells.For example, in RPE cell preparation enriched with 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.Preparation can be substantially purified with respect to non-RPE cells and enriched with respect to differentiated RPE cells but 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.

[0271] The preparations described herein may 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 1 and 2, SV40, HHVS, 6, 7, 8, CMV, polyomavirus, HPV, enterovirus). The preparations described herein may be substantially free of mycoplasma contamination or infection.

[0272] Another way to characterize 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.

[0273] 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., between 80-100% of the cells express MITF.

[0274] 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 bestrophin 1, as measured by immunostaining. For example, between 80-100% of the cells co-express MITF and bestrophin 1.

[0275] 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.

[0276] According to other embodiments, at least 80%, 85%, 87%, 89%, 90%, 95%, 97% or 100% of the cells express both Z0-1 and bestrophin 1 as measured by immunostaining.

[0277] For example, 80-100% of the cells co-express Z0-1 and bestrophin 1.

[0278] According to other embodiments, 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%-100% of the cells express paired box gene 6 (PAX-6).

[0279] According to other embodiments, 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.

[0280] According to other embodiments, at least 80%, 85%, 87%, 89%, 90%, 95%, 97% or 100% of the cells express the cell melanocyte lineage-specific antigen GP100 (PMEL17) as measured by immunostaining, e.g., about 80-100% of the cells express PMEL17.

[0281] The 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 of the resulting RPE cell population co-express both pre-melanosome protein (PMEL17) and cellular retinaldehyde-binding protein (CRALBP).

[0282] According to certain embodiments, 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).

[0283] According to certain embodiments, at least 80% of the cells of the population express detectable levels of PMEL17 and one of the above-mentioned 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-mentioned 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-mentioned 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-mentioned polypeptides (e.g., CRALBP), and more preferably 100% of the cells of the population express detectable levels of PMEL17 and one of the above-mentioned polypeptides (e.g., FACS).

[0284] According to other embodiments, the level of co-expression (e.g., as measured by mean fluorescence intensity) of CRALBP and one of the above polypeptides (e.g., PMEL17) 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.

[0285] 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.

[0286] The RPE cells described herein can also act as functional RPE cells after transplantation, where the RPE cells can form a monolayer between the neurosensory retina and the choroid of the patient receiving the transplanted cells. The RPE cells can also provide nutrients to adjacent photoreceptor cells and dispose of shed photoreceptor outer segments by phagocytosis.

[0287] According to one embodiment, the transepithelial electrical resistance of the cells in the monolayer is greater than 100 ohms.

[0288] 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. The resistance can be any value or subrange within the recited ranges, including the endpoints.

[0289] Devices for measuring transepithelial electrical resistance (TEER) are known in the art and include, for example, the EVOM2 epithelial plethysmometer (World Precision Instruments).

[0290] After the proliferation phase, a cell population is obtained comprising RPE cells, 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% of which are CRALBP+PMEL17+.

[0291] Those skilled in the art will fully understand that the induction of RPE cells brings great benefits. They can be used as an in vitro model for the development of new drugs to 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 reveal mechanisms, new genes, soluble or membrane-bound factors that are important for the development, differentiation, maintenance, survival and function of photoreceptor cells.

[0292] The RPE cells described herein may also serve as an unlimited source of RPE cells for transplantation, replacement and support of dysfunctional or degenerated RPE cells in retinal degeneration and other degenerative disorders.Furthermore, genetically modified RPE cells may function as vectors for delivery and expression of genes in the eye and retina after transplantation.

[0293] In certain embodiments, RPE cell compositions may be produced according to the following method: (1) culturing hESCs on hUCF in CW plates for 2 weeks in NUT+ with human serum albumin (HSA), (2) mechanically passaging to expand hESCs on hUCF in CW plates for 4-5 weeks (or until desired amount of cells) in NUT+ with HSA, (3) continuing to grow hESC colonies (e.g., using collagenase) on hUCF in 6 cm plates for an additional week in NUT+ with HSA, (4) culturing hESCs from approximately 15 6 cm plates in NUT- with nicotinamide (NIC), and (5) culturing hESCs from approximately 15 6 cm plates in NUT- with nicotinamide (NIC). (5) prepare spheroid bodies (SB) by transferring colonies to HydroCell for about 1 week; (6) culture adherent cells on Lam511 in NUT- with NIC for about 1 week by transferring SB to 2-3 wells of a 6-well plate; (7) enrich for pigment cells using enzymes, such as TrypLE Select; (8) grow RPE cells on gelatin in flasks in 20% human serum and NUT- for about 2-9 weeks (replace medium); (9) harvest RPE cells.

[0294] Harvesting of the expanded population of RPE cells can be performed using methods known in the art (e.g., using an enzyme 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 prior to formulation of the RPE cell composition for cryopreservation and administration to a subject immediately after thawing. In some embodiments, the percent viability of the cells after filtration is at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%. In some embodiments, the percent viability of the cells after filtration stored in the neutralizing solution for about 0 to about 8 hours is at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%.

[0295] In further embodiments, the percent viability of cells after filtration following storage in neutralizing medium for about 0 to about 8 hours followed by storage in cryopreservation medium for about 0 to about 8 hours is at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. In other embodiments, the percent recovery of cells after filtration following storage in neutralizing medium for about 0 to about 8 hours followed by storage in cryopreservation medium for about 0 to about 8 hours is at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%.

[0296] In yet other embodiments, the percent viability of cells after filtration after storage in neutralizing medium for about 0 to about 8 hours, followed by storage in cryopreservation medium for about 0 to about 8 hours, and thawing the cryopreservation composition is at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. In yet other embodiments, the percent recovery of cells after filtration after storage in neutralizing medium for about 0 to about 8 hours, followed by storage in cryopreservation medium for about 0 to about 8 hours, and thawing the cryopreservation composition is at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%.

[0297] In some embodiments, filtered RPE cells after storage in neutralizing medium for about 0 to about 8 hours, followed by storage in cryopreservation medium for about 0 to about 8 hours, and thawing the cryopreservation composition may secrete about 1,500 ng / ml / day to about 4,500 ng / ml / day, about 2,000 ng / ml / day to about 3,000 ng / ml / day of PEDF. The concentration may be any value or subrange within the recited range, including the endpoint. In other embodiments, filtered RPE cells after storage in neutralizing medium for about 0 to about 8 hours, followed by storage in cryopreservation medium for about 0 to about 8 hours, and thawing the cryopreservation composition may secrete at least about 1.2x10 PEDF over 14 days. 6 ~5x10 6 , or about 2.5x10 6 ~about 4x10 6 The cells can be expanded to 100 cells.

[0298] In some embodiments, the percent viability of filtered RPE cells stored in neutralizing medium at room temperature for about 0 to about 8 hours is at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%. In some embodiments, the percent viability of filtered RPE cells stored in cryopreservation medium at room temperature for about 0 to about 8 hours is at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%. In further embodiments, the percent viability of cells after filtration following storage in neutralizing solution at room temperature for about 0 to about 8 hours followed by storage in cryopreservation medium at room temperature for about 0 to about 8 hours is at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. In still further embodiments, the percent recovery of cells after filtration following storage in neutralizing solution at room temperature for about 0 to about 8 hours followed by storage in cryopreservation medium at room temperature for about 0 to about 8 hours is at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 105%, 110%, 115%, 120%, 125%, 130%, 140%, 150%.

[0299] After harvesting, the expanded population of RPE cells can be formulated in a specific therapeutic dose (e.g., cell number) and cryopreserved for transport to clinic. Ready-to-administer (RTA) RPE cell therapy composition can then be administered directly after thawing without further processing. 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.

[0300] RPE cells formulated in a cryopreservation medium suitable for ready-to-thaw (RTA) use may 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, dimethylsulfoxide (DMSO), and RPE cells suspended in water. One example of this cryopreservation medium is commercially available under the trade name CRYOSTOR® (e.g., CRYOSTOR® 5) and manufactured by BioLife Solutions, Inc.

[0301] In further embodiments, the cryopreservation medium comprises a purine nucleoside (e.g., adenosine), a branched glucan (e.g., dextran 40), a zwitterionic organic chemical buffer (e.g., HEPES (N-(2-hydroxyethyl)piperazineEN'-(2Eethanesulfonic acid))), and a polar aprotic solvent that the cells can tolerate (e.g., dimethylsulfoxide (DMSO). In still further embodiments, one or more of the purine nucleoside, branched glucan, buffer, and polar aprotic solvent are generally recognized as safe by the U.S. FDA.

[0302] 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.

[0303] In other embodiments, one or more of the sugar acids, bases, halide salts, basic salts, antioxidants, phosphates, sugars, sugar alcohols are generally recognized as safe by the U.S. FDA.

[0304] DMSO may be used as a cryoprotectant to prevent the formation of ice crystals that may kill cells during the cryopreservation process. In some embodiments, the cryopreservable RPE cell therapy composition comprises about 0.1% to about 2% DMSO (v / v). In some embodiments, the RTA RPE cell therapy composition comprises about 1% to about 20% DMSO. In some embodiments, the RTA RPE cell therapy composition comprises about 2% DMSO. In some embodiments, the RTA RPE cell therapy composition comprises about 5% DMSO.

[0305] In some embodiments, the RPE cell therapy formulated in a cryopreservation medium suitable for use immediately after thawing may include RPE cells suspended in a cryopreservation medium without DMSO. For example, the RTA RPE cell therapy composition may include RPE cells suspended in Trolox, Na+, K+, Ca2+, Mg2+, cl-, H2P04-HEPES, lactobionic acid, 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, also manufactured by BioLife Solutions, Inc. In other embodiments, the RPE cell composition formulated in a cryopreservation medium suitable for use immediately after thawing may include RPE cells suspended in trehalose.

[0306] The RTA RPE cell therapy formulated according to the present disclosure does not require the use of GMP facilities to prepare final dose formulations before injection into the eye of a subject.The RTA RPE cell therapy formulations described herein can be stored frozen in a non-toxic freezing solution, including final dose formulations that can be shipped directly to clinical sites.When necessary, the formulations can be thawed and administered to the eye of a subject without the need to carry out intermediate preparation steps.

[0307] In some embodiments, the RPE cell composition may be cryopreserved and stored at a temperature of about -4°C to about -200°C. In some embodiments, the RPE cell composition may be cryopreserved and stored at a temperature of about -20°C to about -200°C. In some embodiments, the RPE cell composition may 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, -70°C to about -196°C.

[0308] In some embodiments, the RTA RPE cell therapy composition may be cryopreserved for about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31 days. In other embodiments, the RPE cells may be cryopreserved for about 1.5 to 48 months. In other embodiments, the RTA RPE cell therapy composition may be cryopreserved for about 1 to about 48 months without loss of viability or cell recovery. In some embodiments, the RTA RPE cell therapy composition may be stored at 2 to 8°C for at least about 38 hours while maintaining stability.

[0309] In some embodiments, RTA RPE cell therapy compositions can be shipped frozen over 8,000 miles without compromising viability, cell recovery or potency.

[0310] RPE cells may be produced, for example, according to the method 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.

[0311] The RTA RPE cell therapy composition may optionally include additional factors that support RPE engraftment, integration, survival, efficacy, and the like. 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. The concentrations can be any value or subrange within the recited ranges, including the endpoints.

[0312] In some embodiments, the RTA RPE cell therapy composition may be formulated to include activators of various integrins that have been shown to increase adhesion to branch membranes of RPE cell preparations, such as those described herein. 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.

[0313] In other embodiments, the RTA RPE cell therapy composition may also be formulated to include an activator of RPE cell immunomodulatory activity.

[0314] In some embodiments, the RTA RPE cell therapy composition may include a ROCK inhibitor.

[0315] In some embodiments, the RTA RPE cell therapy composition may be formulated in a medium containing components that reduce molecular cellular stress during the freezing and thawing process by scavenging free radicals, pH buffering, cancer / osmotic support, and maintaining ion concentration balance.

[0316] In some embodiments, RPE cell therapy formulated in a cryopreservation medium suitable for use in a ready-to-use application after thawing may include one or more immunosuppressant compounds. In certain embodiments, RPE cell therapy formulated in a cryopreservation medium suitable for use in a ready-to-use application 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, mycophenolic acid and small molecule 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, RITUXUMAB® (anti-CD20 antibody), sirolimus, tacrolimus, tacrolimus and / or mycophenolate mofetil.

[0317] Further methods for producing RPE cells contemplated within the present disclosure are described in PCT / US2018 / 023030 (International Publication No. 2018 / 170494), the contents of which are incorporated by reference in their entirety herein.

[0318] Further methods for making the "thaw-inject" formulations contemplated within the present disclosure are described in PCT / IB2018 / 001579 (WO 2019 / 130061), the contents of which are incorporated herein by reference in their entirety.

[0319] 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 ... The cells may be formulated at a cell concentration of 1,000,000 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. The cells may be any value or subrange within the recited ranges, including the endpoints.

[0320] In some embodiments, the RPE cells are administered in a therapeutically or pharma- ceutically acceptable carrier or biocompatible medium. In some embodiments, the volume of the RPE formulation administered to the 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, two or more doses of the RPE formulation of 10 μl to 200 μl may be administered. In certain embodiments, a volume of the RPE formulation is administered to the subretinal space of the subject's eye. In certain embodiments, the subretinal delivery method may be transvitreal or suprachoroidal. In some embodiments, for some subjects, transvitreal or suprachoroidal subretinal delivery methods may be used to reduce the incidence of ERM. In some embodiments, a volume of the RPE formulation may be injected into the subject's eye.

[0321] In some embodiments, the RPE cells of the cellular therapeutic agent are human RPE cells.

[0322] In some embodiments, the RPE cells are OpRegen® cells. OpRegen is an RPE cell line derived from a human embryonic stem cell line (hESC), which is enriched for RPE population by culturing in low oxygen (5%) with high concentrations of Activin A, transforming growth factor beta (TGF-b) family and nicotinamide, then switching to normal oxygen (20%) culture. Activin A improves the survival of RPE cells on stiff or hard substrates, but not on soft substrates. Thus, OpRegen has acquired additional biological capabilities compared to native RPE cells, and enhanced survival in harsh microenvironments, such as GA settings, where Bruch's membrane degenerates and becomes stiff or thickened. Among the more than 120+ identified proteins secreted by OpRegen cells, pigment epithelium-derived factor (PEDF), platelet-derived growth factor (PDGF), vascular endothelial growth factor (VEGF), bestrophin, angiogenin, CRLABP, TIMP-2, TIMP-1, IL-6, PMEL-1 (melonosome), integrins, TNF-α and complement protection proteins top the list as highly secreted proteins. Its efficacy was tested by basal PEDF / VEGF ratio and apical VEGF / PEDF ratio on day 21, both of which were >1. Notably, high oxygen levels increase PEDF secretion. OpRegen in suspension formulation can still produce PEDF for 24 hours at 2-8°C, indicating its robustness.

[0323] OpRegen secretes very high levels of PEDF at 2000-4000ng / ml / day, which may explain its high therapeutic efficacy, since PEDF has an antioxidant role in RPE against BRB, which is of interest for AMD indications. PEDF is a 50kDa protein secreted by RPE and Müller glia in vivo; it also demonstrates a neuroprotective function for photoreceptor cells, possibly by restoring mitochondrial dynamics that are disturbed by aging and oxidative stress. PEDF could prevent H202-induced RPE permeability changes and maintain the barrier function of RPE against oxidative stress. PEDF is also an endogenous anti-inflammatory factor through interaction with the master factor NF-KappaB. PEDF binds to the extracellular matrix (collagen and proteoglycans) and plays a role in anti-fibrosis in diabetic retinopathy and wet AMD through inhibition of TGF-beta. In part, PEDF secretion corroborates findings in OpRegen-treated subjects, as evidenced by improved fluorescein angiograms (FA) in patients with and without drusen, and OCT imaging with possible signs of ECM remodeling or scar attenuation within GA lesions seen as early as 2-4 weeks post-implantation.

[0324] RPE cells suitable for use within the scope of the present disclosure are not limited to the RPE cells described herein Any commercially available or otherwise available RPE cells may be used.

[0325] In some embodiments, the cellular therapeutics described herein can restore retinal structure in retinal diseases.

[0326] Restoration of a patient's retinal anatomy may be used interchangeably with "restoration" and "restoring" and means restoration or regeneration of normal anatomy in a patient compared to an age-matched and gender-matched control, baseline, or fellow eye; restoration of areas of normal anatomy as determined by changes in the ellipsoid zone (EZ) in the affected area, RPE engraftment as evidenced by OCT, and improved retinal thickness; restoration or induction of retinal pigment epithelium (RPE) regeneration; restoration of areas of normal anatomy as determined by changes in the ellipsoid zone (EZ) in the affected area, RPE engraftment as evidenced by OCT, and improved retinal thickness; restoration of visual acuity; reducing areas of atrophy in an atrophic retina; restoring one or more retinal layers of the retina; restoring photoreceptors of the retina; restoring the outer nuclear layer (ONL) of the retina; restoring the ellipsoid zone (EZ) of the retina; restoring the fovea of ​​the retina; restoring the blood-retinal barrier (BRB) of the retina; and restoring the extracellular matrix (ECM) of the retina.

[0327] Restoring or regenerating the function of a patient's retina means that the retinal layers are restored to their normal structure and that the RPE cells, which carry out activities such as light absorption, epithelial transport, phagocytosis of photoreceptor outer segment (POS) membranes, and secretion of factors such as PEDF and photoreceptors, are functionally active and capable of phototransduction, thereby allowing functional vision.

[0328] "Recovery" and "recover" and "recovers" and "recovering" refer to regeneration of the ellipsoid zone; regeneration with restoration of normal architecture; compared to age-matched and gender-matched controls, baseline or fellow eye; subjective assessment that one or more of the following are becoming more organized, including but not limited to: the external limiting membrane, the myoid region (inner segment of photoreceptors), the ellipsoid zone (IS / OS junction), the outer segments of photoreceptors, loss of drusen, and disappearance of reticular pseudodrusen; ), and a subjective assessment that one or more of the fundamental foundational layers of the retina are becoming more organized, including one or more of the outer segments of the photoreceptor cells; demonstrating that an area of ​​the retina near or at the site of administration of RPE cells contains an improved microperimetric assessment compared to a baseline microperimetric assessment; regeneration of the ellipsoid zone, including improvement in one or more of EZ-RPE thickness, area, or volume measurements; improvement in EZ-RPE foveal mean thickness; improvement in EZ-RPE foveal thickness; improvement in EZ-RPE central subregion volume; regeneration of pigment epithelium and retinal thickness; organization of the fundamental foundational layers of the retina; may be used interchangeably to mean organization of 2-6 of the 12-14 layers of the retina.

[0329] Treatment and Dosage 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 cellular therapy comprises at least about 50,000 viable cells. In some embodiments, the cellular therapy comprises at least about 100,000 viable cells. In some embodiments, the cellular therapy comprises at least about 150,000 viable cells. In some embodiments, the cellular therapy comprises at least about 200,000 viable cells. In some embodiments, the cellular therapy comprises at least about 250,000 viable cells. In some embodiments, the cellular therapy comprises at least about 300,000 viable cells. In some embodiments, the cellular therapy comprises at least about 350,000 viable cells. In some embodiments, the cellular therapy comprises at least about 400,000 viable cells. In some embodiments, the cellular therapy comprises at least about 450,000 viable cells. In some embodiments, the cellular therapy comprises at least about 500,000 viable cells. In some embodiments, the cell therapy 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. In some embodiments, the cell therapy comprises 50,000 to 100,000 viable cells. In some embodiments, the cell therapy comprises 100,000-200,000 viable cells. In some embodiments, the cell therapy comprises 200,000-300,000 viable cells. In some embodiments, the cell therapy comprises 300,000-400,000 viable cells. In some embodiments, the cell therapy comprises 400,000-500,000 viable cells. In some embodiments, the cell therapy comprises 500,000-1,000,000 viable cells.In some embodiments, the cell therapy agent comprises 1,000,000 to 2,000,000 viable cells. In some embodiments, the cell therapy agent comprises 2,000,000 to 3,000,000 viable cells. In some embodiments, the cell therapy agent comprises 3,000,000 to 4,000,000 viable cells. In some embodiments, the cell therapy agent comprises 4,000,000 to 5,000,000 viable cells. In some embodiments, the cell therapy agent comprises 5,000,000 to 6,000,000 viable cells. In some embodiments, the cell therapy agent comprises 6,000,000 to 7,000,000 viable cells. In some embodiments, the cell therapy agent comprises 7,000,000 to 8,000,000 viable cells. In some embodiments, the cellular therapy comprises between 8,000,000 and 9,000,000 viable cells. In some embodiments, the cellular therapy comprises between 9,000,000 and 10,000,000 viable cells. In some embodiments, the cellular therapy comprises between 10,000,000 and 11,000,000 viable cells. In some embodiments, the cellular therapy comprises between 11,000,000 and 12,000,000 viable cells. In certain embodiments, the cellular therapy is administered at a dose of between 50,000 and 1,000,000 cells. In certain embodiments, the cellular therapy is administered at a dose of between 100,000 and 750,000 cells. In certain embodiments, the cellular therapy is administered at a dose of between 200,000 and 500,000 cells. Each of the values ​​or ranges recited herein can include any value or subrange therebetween, including the endpoints.

[0330] In some embodiments, the volume of the RTA RPE formulation administered to the subject is about 50 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, two doses of the RTA RPE formulation of 10 μl to 200 μl may be administered. Each of the values ​​or ranges recited herein may include any value or subrange therebetween, including the endpoints.

[0331] In certain embodiments, a certain amount of RTA RPE formulation is administered to the subretinal space of the eye of the subject.In certain embodiments, the subretinal delivery method can be transvitreal or suprachoroidal.In some embodiments, a certain amount of RTA RPE formulation can be injected into the eye of the subject.

[0332] In certain embodiments, RTA RPE therapeutic cell compositions may be formulated at cell concentrations of about 100,000 cells / ml to about 1,000,000 cells / ml. In certain embodiments, RTA RPE cell therapy may be formulated at cell concentrations 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, about 11,000 cells / ml, or about 12,000 cells / ml. The cell concentration may be formulated at about 10,000 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. Each of the values ​​or ranges recited herein may include any value or subrange therebetween, including the endpoints.

[0333] In some embodiments, the method comprises administering RPE cells to the subject's eye. In some embodiments, the method comprises administering RPE cells to the subretinal space of the subject's eye. In some embodiments, the method comprises administering RPE cells to the vitreous space, the inner or outer retina, the periretina, or the choroid of the subject's eye. In some embodiments, the method comprises administering RPE cells onto a GA lesion. In some embodiments, the method comprises targeting GA in the subject's eye. In some embodiments, the method comprises administering RPE cells by lifting the GA. In some embodiments, the method comprises administering RPE cells to the surrounding healthy tissue near the GA lesion. In some embodiments, the RPE cells are administered as a monolayer. In some embodiments, the cell composition is injected.

[0334] The RPE cells generated as described herein can be transplanted into various target sites in the eye of a subject or elsewhere (e.g., in the brain). According to one embodiment, the transplantation of RPE cells is into the subretinal space of the eye, which is the normal anatomical location of RPE (between the photoreceptor outer segment and the choroid). Furthermore, depending on the migratory ability and / or positive paracrine action of the cells, transplantation into additional ocular compartments, including but not limited to the vitreous space, the inner and outer retina, the periretina, and the choroid, can be considered.

[0335] Transplantation can be carried out by various techniques known in the art.The method of carrying out RPE transplantation is described in, for example, U.S. Patent No. 5,962,027, No. 6,045,791 and No. 5,941,250, and Eye Graefes Arch Clin Exp Opthalmol March 1997;235(3):149-58;Biochem Biophys Res Commun Feb.24,2000;268(3):842-6;Opthalmic Surg February 1991;22(2):102-8. Methods for performing corneal transplantation 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 mainly utilizing the paracrine effect, cells can also be delivered and maintained in the eye encapsulated in a semi-permeable container or a biodegradable extracellular matrix, which also reduces the exposure of cells to the host immune system (Neurotech USA CNTF delivery system;PNAS March 7,2006 vol.103(10)3896-3901).

[0336] In some embodiments, the cellular therapeutic agent is administered adjacent to the GA.

[0337] In some embodiments, the cellular therapeutic agent is administered adjacent to the GA. In some embodiments, the cellular therapeutic agent is administered to the GA. In some embodiments, the cellular therapeutic agent covers at least about 20% of the GA after administration. In some embodiments, the cellular therapeutic agent covers at least about 30% of the GA after administration. In some embodiments, the cellular therapeutic agent covers at least about 40% of the GA after administration. In some embodiments, the cellular therapeutic agent covers at least about 50% of the GA after administration. In some embodiments, the cellular therapeutic agent covers at least about 60% of the GA after administration. In some embodiments, the cellular therapeutic agent covers at least about 70% of the GA after administration. In some embodiments, the cellular therapeutic agent covers at least about 75% of the GA after administration. In some embodiments, the cellular therapeutic agent covers at least about 80% of the GA after administration. In some embodiments, the cellular therapeutic agent covers at least about 85% of the GA after administration. In some embodiments, the cellular therapeutic agent covers at least about 90% of the GA after administration. In some embodiments, the cellular therapeutic agent covers at least about 95% of the GA after administration. In some embodiments, the cellular therapeutic agent covers at least about 96% of the GA after administration. In some embodiments, the cellular therapeutic agent covers at least about 97% of the GA after administration. In some embodiments, the cellular therapeutic agent covers at least about 98% of the GA after administration. In some embodiments, the cellular therapeutic agent covers at least about 99% of the GA after administration. In some embodiments, the cellular therapeutic agent covers about 100% of the GA after administration.

[0338] According to one embodiment, transplantation is performed via a partial planar vitrectomy surgery followed by delivery of the cells to the subretinal space through a small retinal opening or by direct injection.

[0339] In certain embodiments, administration may involve vitrectomy followed by delivery of the RTA therapeutic cell composition via a cannula through a small retinotomy into the subretinal space in the macular region. Depending on the cell dose, a total volume of 50-100 µL of cell suspension may be implanted into areas at potential risk for GA enlargement.

[0340] In some embodiments, a single surgical procedure is performed in which the RTA therapeutic cell composition is delivered through a small retinotomy after vitrectomy into the subretinal space formed in the macular region along the border between the area of ​​GA and the better preserved extrafoveal retina and RPE layers, if present. After placement of the lid speculum, a standard three-port vitrectomy may be performed. This may include placement of a 23G or 25G injection cannula and two 23G or 25 / 23G ports (trocars). A core vitrectomy may then be performed with a 23G or 25G instrument, followed by peeling of the posterior vitreous face. The RTA therapeutic cell composition may be injected into the subretinal space at a predetermined site within the posterior pole, preferably penetrating the retina in an area that is still relatively preserved near the border of GA, if present.

[0341] In some embodiments, the cell composition is administered by suprachoroidal injection.

[0342] 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 with a matrix, or can be attached to a matrix or membrane, extracellular matrix or substrate, such as a biodegradable polymer or combination. 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 the treatment can be evaluated by various measures of visual and ocular function and structure, in particular 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, adoptive optics, and additional measures used to evaluate visual function and ocular structure.

[0343] The subject may be administered a corticosteroid, such as prednisolone or methylprednisolone, predforte, prior to or concurrently with administration of the RPE cells. According to other embodiments, the subject is not administered a corticosteroid, such as prednisolone or methylprednisolone, predforte, prior to or concurrently with administration of the RPE cells.

[0344] Immunosuppressants can be administered to subjects before, at the same time and / or after treatment.Immunosuppressants can belong to the following classes: glucocorticoids, cytostatics (e.g., alkylating agents or antimetabolites), antibodies (polyclonal or monoclonal), drugs that act on immunophilins (e.g., cyclosporine, tacrolimus or sirolimus).Additional drugs include interferons, opioids, TNF-binding proteins, mycophenolic acid and small molecule biological agents. Examples of immunosuppressants include mesenchymal stem cells, antilymphocyte globulin (ALG) polyclonal antibody, antithymocyte globulin (ATG) polyclonal antibody, azathioprine, BAS 1L1 X 1MABO (anti-IL-2Ra receptor antibody), cyclosporine (cyclosporine A), DACLIZUMAB® (anti-IL-2Ra receptor antibody), everolimus, mycophenolic acid, RITUX 1MABO (anti-CD20 antibody), sirolimus, tacrolimus, tacrolimus and / or mycophenolate mofetil.

[0345] Immunosuppressants can be administered to a subject, for example, topically, intraocularly, intraretinaly, or systemically. Immunosuppressants can be administered by one or more of these methods simultaneously, or delivery methods can be used in a staggered fashion.

[0346] Alternatively, the RTA RPE cell therapy composition may be administered without the use of immunosuppressants.

[0347] Antibiotics may be administered to the subject before, during 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.

[0348] In some embodiments, the cell composition does not cause inflammation following administration, hi some embodiments, inflammation may be characterized by the presence of cells associated with inflammation.

[0349] In some embodiments, recovery results in a reduction in the area of ​​atrophy. At a specified time after treatment, fundus autofluorescence (FAF) can be used to detect any hyperfluorescence, especially around the edge of the lesion, and measure the size of the area of ​​atrophy. In addition to a reduction in the overall size of the lesion, a reduction in the size or disappearance of the hyperfluorescent rim around the lesion can be used to indicate that the treatment is slowing or halting the progression of the disease. The difference in hyperfluorescence between the treated half of the lesion and the untreated half of the lesion can be measured and used to determine the effectiveness of the treatment. Thus, the same eye can be used as the treated and control subjects.

[0350] In some embodiments, the recovery results in a decrease in the atrophic area. As used herein, the terms "decrease", "reduce", "reduction", "minimal", "low" or "lower" refer to a decrease below basal levels, e.g., compared to a control. The terms "increase", "high", "higher", "maximal", "elevate" or "elevation" refer to an increase above basal levels, e.g., compared to a control. Increase, elevation, decrease, or decline is 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109 ... 9%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. Each of the values ​​or ranges recited herein can include any value or subrange therebetween, including the endpoints.

[0351] In certain embodiments, the treatment results in the recovery of the retinal layers. In other embodiments, treatment efficacy assessment using two-dimensional images of fundus autofluorescence is enhanced using optical coherence tomography (OCT). OCT can be used to generate three-dimensional high-resolution images and can provide important cross-sectional information for structural assessment of the retinal layers, especially in subjects undergoing treatment for retinal disease. Using OCT, profile images of the layers of the retina can be obtained before and after treatment for retinal disorders is administered. In healthy eyes, the individual layers of retinal tissue can be seen as distinct bands. Conversely, characteristic defects caused, for example, by AMD or GA can be seen as clearly defined areas of deterioration in the RPE and photoreceptor layers. In many eyes with GA, OCT images can show wedge-shaped hyporeflective structures that can develop between the branch membrane and the outer plexiform layer. Identification and monitoring of such structures can be useful in defining the OCT boundaries of the photoreceptor layers, which is important in clinical trials of treatments aimed at maintaining the viability of the retinal layers in patients with AMD and GA.

[0352] By combining segmentation of retinal layers in OCT with metabolic mapping of fundus autofluorescence, morphological changes associated with functional changes can be seen more clearly. Using specialized software, the lesion area seen in the FAF images can be quantified and tracked over time. Treatment effects can also be identified, including areas of RPE regeneration overlying the lesion, and RPE recovery can be quantified by measuring retinal thickness.

[0353] In some embodiments, the treatment results in the recovery of photoreceptors. RPE cells are involved in many processes important for photoreceptor survival, including nutrient, water and ion transport, light absorption, phagocytosis of shed photoreceptor outer segments (POS), and reisomerization of all-trans retinal to 11-cis retinal, which is important for the visual cycle, immune regulation, secretion of essential factors and formation of the blood-retinal barrier. The RPE monolayer acts as a polarized metabolic gatekeeper between the PR and the choriocapillaris (CC). The RPE has structural and functional polarity from apical to basal. At the apical side, RPE cells form multiple villi that allow direct contact with POS and transport molecules such as glucose and vitamin A from the choriocapillaris to the PR. At the basal side, RPE cells generate the underlying basement Bruch's membrane (BM) that separates the RPE from the choroid, which transports metabolic products such as CO2, lactate and water to the choriocapillaris and creates the blood-retinal barrier. At the lateral wall, adjacent RPE cells form tight junctions. Barrier function can be used to determine the potency of RPE cell cultures by measuring the tight junctions formed between cells. RPE tight junctions limit the paracellular movement of ions and water across the RPE monolayer and maintain the correct apical-basal distribution of RPE transporters. The RPE cell compositions disclosed herein can be cultured at a resistance of 100 Ω (100 Ω*cm 2 ) to generate a transepithelial electrical resistance (TEER) greater than 0.01.

[0354] In addition, RPE cells secrete various neurotrophic factors, such as fibroblast growth factors (bFGF and aFGF), ciliary neurotrophic factor (CNTF), pigment epithelium-derived factor (PEDF), brain-derived neurotrophic factor (BDNF), vascular endothelial growth factor (VEGF), etc., that help maintain the structural integrity of ciliary capillary endothelium and photoreceptors. RPE cells also secrete anti-inflammatory cytokines, such as transforming growth factor (TGF)-β, which are important for establishing the immune privilege characteristics of the eye. The RPE cells used in the RTA therapeutic cell composition described herein can secrete neurotrophic factors. The RPE cell composition disclosed herein also exhibits polarized PEDF and VEGF secretion, which enhances RPE proliferation and angiogenesis, respectively.

[0355] In certain embodiments, RPE cell grafts provide long-lasting trophic support for degenerating retinal tissue by secreting these factors after transplantation. This trophic support is some of the targets that can act to attenuate retinal degradation and vision loss. Trophic factors are known as cell survival and differentiation promoters. Examples of trophic factors and trophic factor families include, but are not limited to, neurotrophins, ciliary neurotrophic factor / leukemia inhibitory factor (CNTF / LIF) family, hepatocyte growth factor / scatter factor family, insulin-like growth factor (IGF) family and glial cell line-derived neurotrophic factor (GDNF) family. RPE cells described herein can start secreting trophic factors immediately after administration or retinal transplantation. Furthermore, a steady flow of neuroprotective support can begin once the cells are integrated between recipient cells and establish synaptic contacts with the cells of interest.

[0356] In some embodiments, treatment / administration of RPE cells results in a pluripotent secretory effect of RPE cells, as described by J. Cell. Mol. Med. Vol 17, No 7, 2013 pp. 833-843, the entire contents of which are incorporated herein by reference.

[0357] In some embodiments, treatment can result in the recovery of the outer nuclear layer (ONL).ONL (or outer nuclear layer or outer nuclear layer) is one of the layers of the vertebrate retina, which is the light-detecting part of the eye.Similar to the inner nuclear layer, the outer nuclear layer contains several layers of ellipsoidal nuclei, two types of which are named rod nuclei and cone nuclei because they are connected with the rods and cones of the next layer, respectively.

[0358] The spherical rod granules are much more numerous and are arranged at different levels throughout the layer. Their nuclei have a peculiar transversely striped appearance, and extending from either end of each cell are fine processes. The outer process is continuous with a single rod in the rod and cone layer; the inner end lies in the outer plexiform layer at the enlarged end and is embedded in a tuft into which the outer processes of the rod bipolar cells divide. In the process, it presents numerous varicosities.

[0359] The stalk-like cone granules, which are less numerous than the rod granules, are located near the external limiting membrane and through it are continuous with the cones of the rod and cone layers. They do not show cross-detachment, but contain a pyriform nucleus that almost completely fills the cell. From the inner end of the granules, a thick process enters the external plexiform layer, from which it expands into the cone extension or foot plate, from which numerous fine fibrils are released and come into contact with the external process of the cone bipolar.

[0360] In some embodiments, treatment may result in restoration of the ellipsoid zone, as described elsewhere herein.

[0361] In some embodiments, treatment may result in restoration of the fovea of ​​the retina.

[0362] In some embodiments, treatment may result in restoration or repair of the blood-retinal barrier (BRB), as described elsewhere herein.

[0363] In some embodiments, the recovery may result in remodeling of the extracellular matrix (ECM). The ECM is a three-dimensional network of extracellular polymers and minerals, such as collagen, enzymes, glycoproteins and hydroxyapatite, that provide structural and biochemical support to surrounding cells. Because multicellularity evolved independently in different multicellular lineages, the composition of the ECM differs between multicellular structures, but cell adhesion, cell-cell communication and differentiation are common functions of the ECM.

[0364] Animal extracellular matrix includes interstitial matrix and basement membranes. Interstitial matrix resides between various animal cells (i.e., in the intercellular spaces). A gel of polysaccharides and fibrous proteins fills the interstitial spaces and acts as a compressive buffer against stresses placed on the ECM. Basement membranes are sheet-like deposits of ECM upon which various epithelial cells reside. Each type of connective tissue in animals has a type of ECM: collagen fibers and bone minerals comprise the ECM of bone tissue, reticular fibers and matrix comprise the ECM of loose connective tissue, and plasma is the ECM of blood.

[0365] In some embodiments, recovery includes one or more of: reduced growth of geographic atrophy, improved visual acuity, improved reading speed, improved retinal structure, reduced drusen (waste products removed by RPE cells), or stable engraftment of cells.

[0366] In some embodiments, amelioration includes a decrease in the growth of geographic atrophy. In some embodiments, decreasing the growth of geographic atrophy includes decreasing the size of geographic atrophy, e.g., decreasing the total area of ​​atrophy. In some embodiments, reducing the growth of geographic atrophy includes decreasing the growth of atrophic lesions. In some embodiments, the atrophic lesions are isolated (not related to primary GA). In some embodiments, reducing the growth of geographic atrophy includes decreasing the growth rate of geographic atrophy. In some embodiments, the decrease is compared to a control (e.g., expected growth or growth rate, historical growth or growth rate, growth or growth rate in untreated eyes, average growth or growth rate for subjects with similar disease or disorder, or growth or growth rate in comparable subjects, etc.).

[0367] In some embodiments, the growth of geographic atrophy is less than about 98% of the control. In some embodiments, the growth of geographic atrophy is less than about 95% of the control. In some embodiments, the growth of geographic atrophy is less than about 90% of the control. In some embodiments, the growth of geographic atrophy is less than about 85% of the control. In some embodiments, the growth of geographic atrophy is less than about 80% of the control. In some embodiments, the growth of geographic atrophy is less than about 75% of the control. In some embodiments, the growth of geographic atrophy is less than about 70% of the control. In some embodiments, the growth of geographic atrophy is less than about 65% of the control. In some embodiments, the growth of geographic atrophy is less than about 60% of the control. In some embodiments, the growth of geographic atrophy is less than about 50% of the control. In some embodiments, the growth of geographic atrophy is less than about 40% of the control. In some embodiments, the growth of geographic atrophy is less than about 30% of the control. In some embodiments, the growth of geographic atrophy is less than about 25% of the control. In some embodiments, the growth of geographic atrophy is less than about 20% of the control. In some embodiments, the growth of geographic atrophy is less than about 10% of the control. In some embodiments, the growth of geographic atrophy is between about 1% and about 99% of the control. In some embodiments, the growth of geographic atrophy is between about 10% and about 90% of the control. The values ​​can be any value or subrange within the recited ranges, including the endpoints.

[0368] In some embodiments, recovery includes improved vision. In some embodiments, improved vision includes improvement over a control, such as pre-treatment (baseline) vision. In some embodiments, "improvement" includes less vision loss than expected, e.g., less vision loss than a control, less vision loss than an untreated eye, less vision loss than historical loss rates, less vision loss than the average loss rate for subjects with a similar disease or disorder, etc. In some embodiments, improved vision includes improved overall vision. In some embodiments, improved vision includes improved color vision. In some embodiments, improved vision includes improved peripheral vision. In some embodiments, improved vision includes improved distance vision. In some embodiments, improved vision includes improved vision-specific social functioning. In some embodiments, improved vision includes improved vision-specific mental health. In some embodiments, improved vision includes improved vision-specific dependency.

[0369] In some embodiments, the improvement in vision is at least 5% improved compared to the control. In some embodiments, the improvement in vision is at least 10% improved compared to the control. In some embodiments, the improvement in vision is at least 20% improved compared to the control. In some embodiments, the improvement in vision is at least 25% improved compared to the control. In some embodiments, the improvement in vision is at least 30% improved compared to the control. In some embodiments, the improvement in vision is at least 40% improved compared to the control. In some embodiments, the improvement in vision is at least 50% improved compared to the control. In some embodiments, the improvement in vision is at least 60% improved compared to the control. In some embodiments, the improvement in vision is at least 70% improved compared to the control. In some embodiments, the improvement in vision is at least 80%, 90%, 100% or more improved compared to the control. In some embodiments, the improvement in vision is about 5% to about 500% improved compared to the control. In some embodiments, the improvement in vision is about 5% to about 250% improved compared to the control. In some embodiments, the improvement in vision is about 5% to about 100% improved compared to the control. The improvement can be any value or subrange within the recited ranges, including the endpoints.

[0370] In some embodiments, recovery includes improvement in reading speed. In some embodiments, improvement in reading speed includes improvement against a control, such as pre-treatment (baseline) reading speed. In some embodiments, "improvement" includes lower reading speed loss than expected, e.g., lower reading speed loss than a control, e.g., lower reading speed loss than an untreated eye, lower reading speed loss than the historical loss rate, lower reading speed loss than the average loss rate for subjects with a similar disease or disorder, lower reading speed loss than the loss rate for comparable subjects, etc.

[0371] In some embodiments, the improvement in reading speed is at least 5% compared to the control. In some embodiments, the improvement in reading speed is at least 10% compared to the control. In some embodiments, the improvement in reading speed is at least 20% compared to the control. In some embodiments, the improvement in reading speed is at least 25% compared to the control. In some embodiments, the improvement in reading speed is at least 30% compared to the control. In some embodiments, the improvement in reading speed is at least 40% compared to the control. In some embodiments, the improvement in reading speed is at least 50% compared to the control. In some embodiments, the improvement in reading speed is at least 60% compared to the control. In some embodiments, the improvement in reading speed is at least 70% compared to the control. In some embodiments, the improvement in reading speed is at least 80%, 90%, 100% or more compared to the control. In some embodiments, the improvement in reading speed is about 5% to about 500% compared to the control. In some embodiments, the improvement in reading speed is about 5% to about 250% compared to the control. In embodiments, the read rate is improved by about 5% to about 100% compared to the control. The improvement can be any value or subrange within the recited ranges, including the endpoints.

[0372] In some embodiments, restoring comprises increasing the thickness, preventing loss of thickness, or reducing the rate of loss of thickness of one or more regions of the retina. In some embodiments, restoring comprises increasing the area, preventing loss of area, or reducing the rate of loss of area of ​​one or more regions of the retina. In some embodiments, restoring comprises increasing the volume, preventing loss of volume, or reducing the rate of loss of volume of one or more regions of the retina. In some embodiments, the region of the retina comprises a region adjacent to an atrophic region. In embodiments, the region of the retina may be one or more of the following: total retina, foveal center, fovea, central atrophy or lesion, peripheral atrophy or lesion, multifocal lesion, RPE, external limiting membrane (ELM), outer nuclear layer (ONL), outer plexiform layer (OPL), inner nuclear layer (INL), inner plexiform layer (IPL), ganglion cell layer (GCL), retinal nerve fiber layer (RNFL), internal limiting membrane (ILM), ellipsoid zone (EZ), inner / outer segment (IS / OS) of PR.

[0373] In some embodiments, the thickness, area or volume of the region of the retina is improved by at least 5% compared to the control. In some embodiments, the thickness, area or volume of the region of the retina is improved by at least 10% compared to the control. In some embodiments, the thickness, area or volume of the region of the retina is improved by at least 20% compared to the control. In some embodiments, the thickness, area or volume of the region of the retina is improved by at least 25% compared to the control. In some embodiments, the thickness, area or volume of the region of the retina is improved by at least 30% compared to the control. In some embodiments, the thickness, area or volume of the region of the retina is improved by at least 40% compared to the control. In some embodiments, the thickness, area or volume of the region of the retina is improved by at least 50% compared to the control. In some embodiments, the thickness, area or volume of the region of the retina is improved by at least 60% compared to the control. In some embodiments, the thickness, area or volume of the region of the retina is improved by at least 70% compared to the control. In some embodiments, the thickness, area or volume of the region of the retina is improved by at least 80%, 90%, 100% or more compared to the control. In some embodiments, the thickness, area or volume of the retinal region is improved by about 5% to about 500% compared to a control. In some embodiments, the thickness, area or volume of the retinal region is improved by about 5% to about 250% compared to a control. In some embodiments, the thickness, area or volume of the retinal region is improved by about 5% to about 100% compared to a control. The improvement can be any value or subrange within the recited range, including the endpoints.

[0374] In certain embodiments, treating or slowing the progression of, maintaining or reversing a retinal disease is demonstrated by recovery of visual acuity as assessed by microperimetry. Microperimetry, sometimes called fundus-related perimetry, is a type of visual field testing that uses one of several techniques to create a "retinal sensitivity map" of the amount of light perceived in a specific portion of the retina in people who have lost the ability to fixate on objects or light sources. Recovery of visual acuity as assessed by microperimetry includes a correlation between retinal sensitivity in microperimetry and anatomical changes / defects in the retina compared to baseline, age-matched and gender-matched controls, or the fellow eye of the subject. In certain embodiments, treating or slowing the progression of, maintaining or reversing a retinal disease is demonstrated by recovery of visual acuity as assessed by microperimetry, with a correlation between anatomical retinal changes or areas of atrophy as seen by Spectral Domain Optical Coherence Tomography (SD-OCT) and retinal sensitivity loss in 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.

[0375] In other embodiments, to demonstrate the treatment or slowing, maintaining or reversing the progression of retinal disease by comparing the map with age-matched and sex-matched controls, baseline of the subject or fellow eye of the subject, the topography map of the ellipsoid zone, for example, the orthogonal topography (en face) map, is generated from OCT volume scan, for example, Heidelberg Spectralis OCT volume scan (15×10° area, B-scan interval of 30 μm) or Zeiss Cirrus HD-OCT 4000 512×128 cube scan. There is a correlation between the organization of EZ and retinal sensitivity. After administration of RPE cells, the EZ zone is organized and retinal sensitivity is improved. 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., incorporated by reference in its entirety.

[0376] In certain embodiments, treating or slowing, maintaining stasis, or reversing the progression of retinal disease is demonstrated by OCT-A compared to age-matched and gender-matched controls, baseline or fellow eye of the subject before and after administration.

[0377] For example, using spectral domain (SD)-OCT and OCT-A imaging, the 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 may be measured, e.g., using the Optovue Avanti split-spectrum amplitude decorrelation angiography algorithm. EZ-RPE parameters are compared to age-matched and gender-matched controls, the subject's baseline or the fellow eye.

[0378] In one embodiment, after administration, EZ-RPE foveal mean thickness improves, EZ-RPE foveal thickness improves, and EZ-RPE central subregion volume improves. EZ-RPE thickness, area, and volume are correlated with improved visual acuity to measure treatment response. Each of these measurements is inversely correlated with visual acuity. See, for example, the methodology 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 by reference in its entirety.

[0379] In one embodiment, regeneration is a subjective assessment that one or more of the following are becoming more organized: the external limiting membrane, the myoid region (the inner segment of the photoreceptor), the ellipsoid zone (IS / OS junction), the outer segment of the photoreceptor, loss of drusen, and disappearance of reticular pseudodrusen. Regeneration can also include a subjective assessment that one or more of the basic foundational layers of the retina are becoming more organized. As used herein, the basic foundational layers of the retina that become more organized include one or more of the external limiting membrane, the myoid region (the inner segment of the photoreceptor), the ellipsoid zone (IS / OS junction), and the outer segment of the photoreceptor.

[0380] In one embodiment, analysis of the ellipsoid zone demonstrates organization of the EZ by a reduction in EZ volume compared to age-matched and sex-matched controls, baseline or fellow eyes. In other embodiments, the reduction in EZ volume includes 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%. In other embodiments, organization of the EZ is demonstrated, for example, by a reduction in the volume of EZ structures, see, for example, comparison of baseline to months 2 and 3. For example, the volume of the EZ is reduced by at least 2%, at least 5%, at least 10%. Each of the values ​​or ranges recited herein may include any value or subrange therebetween, including the endpoints.

[0381] In one embodiment, regeneration comprises one or more of the following: improvement of EZ-RPE foveal mean thickness, improvement of EZ-RPE foveal thickness, and improvement of EZ-RPE central subregion volume. EZ-RPE thickness, area, and volume are correlated with improvement of visual acuity to measure treatment response. Each of these measurements is inversely correlated with visual acuity.

[0382] In some embodiments, improvement or recovery is measured by microperimetry.

[0383] In microperimetry, specific areas of the retina are stimulated with a light spot and the subject presses a button to confirm perception of the stimulus. In addition to identifying functional and non-functional areas, the stimulus intensity may be varied to identify the relative sensitivity of specific areas of the retina. The fundus may be monitored via an infrared camera and the sensitivity of the visual field may be mapped onto fundus photographs and compared to images obtained with other techniques.

[0384] In certain embodiments, treating or slowing the progression of, maintaining or reversing a retinal disease is demonstrated by restoration of visual acuity as assessed by microperimetry, where restoration of visual acuity as assessed by microperimetry includes a correlation between retinal sensitivity and anatomical changes / defects in the retina as measured by microperimetry, as compared to baseline, an age-matched and gender-matched control, or the fellow eye of the subject. In certain embodiments, treating or slowing the progression of, maintaining or reversing a retinal disease is demonstrated by restoration of visual acuity as assessed by microperimetry, where there is a correlation between anatomical retinal changes or areas of atrophy as seen by Spectral Domain Optical Coherence Tomography (SD-OCT) and retinal sensitivity loss as measured by 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.

[0385] 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 with a matrix, or can be attached to a matrix or membrane, extracellular matrix or substrate, such as a biodegradable polymer or combination. 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 the treatment can be evaluated by various measures of visual and ocular function and structure, in particular 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, adoptive optics, and additional measures used to evaluate visual function and ocular structure.

[0386] In some embodiments, the cell therapy agent is implanted into the subretinal space using a delivery device. In some embodiments, the delivery device comprises a needle, a capillary, and a tip. In some embodiments, the delivery device comprises a needle having an outer diameter of about 0.63 mm and an inner diameter of about 0.53 mm, a capillary having an outer diameter of about 0.5 mm and an inner diameter of about 0.25 mm, and a tip having an outer diameter of about 0.12 mm and an inner diameter of about 0.07 mm.

[0387] In another aspect, there is provided a method of assessing the progression of a retinal disease or disorder as described, illustrated or exemplified herein.

[0388] In one aspect, a method of producing a cellular therapy as described, illustrated or exemplified herein is provided.

[0389] In one aspect, a method is provided for assessing and improving vision according to a rating scale described, illustrated or illustrated herein. In embodiments, the assessment is one or more of the following: reduced growth of geographic atrophy, visual acuity, reading speed, retinal structure, reduced drusen, or stable engraftment of cells. In embodiments, the assessment is reduced growth of geographic atrophy. In embodiments, the assessment is visual acuity. In embodiments, the assessment is reading speed. In embodiments, the assessment is retinal structure. In embodiments, the assessment is reduced drusen. In embodiments, the assessment is stable engraftment of cells.

[0390] For the methods provided herein, in some embodiments, the methods result in minimal or no delayed inflammation of rejection of transplanted cells. In some embodiments, the methods result in minimal rejection of transplanted cells. In some embodiments, the methods result in delayed inflammation of rejection of transplanted cells.

[0391] For the methods provided herein, in some embodiments, the methods include a patient population, patient characteristics, or patient demographics as described, illustrated, or exemplified herein. In some embodiments, the methods include a patient population as described, illustrated, or exemplified herein. In some embodiments, the methods include a patient characteristic as described, illustrated, or exemplified herein. In some embodiments, the methods include a patient demographics as described, illustrated, or exemplified herein.

[0392] In some embodiments, the method may further comprise selecting a patient (subject), a patient population, a patient characteristic, or a patient demographic as described, explained, or exemplified herein.In some embodiments, the patient population suffers from a retinal disease origin or a retinal disease associated with RPE damage, dysfunction, or loss from various pathologies.In some embodiments, the patient population suffers from a retinal disease condition selected from the group consisting of dry AMD, retinitis pigmentosa, Uscher syndrome, vitelliform maculopathy, Stargardt disease, retinal detachment, retinal dysplasia, retinal atrophy, retinopathy, macular dystrophy, cone dystrophy, cone-rod dystrophy, honeycomb retinal dystrophy (Malattia Leventinese), Doyne honeycomb dystrophy, Sorsby dystrophy, pattern / butterfly dystrophy, Best disease, North Carolina dystrophy, central ring choroidal dystrophy, pigmented streaks, toxic maculopathy, pathological myopia, retinitis pigmentosa, and macular degeneration. In some embodiments, the patient is selected to have AMD. In some embodiments, the patient has dry AMD. In some embodiments, the patient has wet AMD.

[0393] In addition to the above-mentioned diseases, the non-limiting list of diseases that can measure the effect of treatment according to the described method also includes 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 dystrophies of RPE, RPE and retina damage caused by any one of light, laser, inflammation, infection, radiation, angiogenesis or traumatic injury.According to certain embodiments, the disease is dry AMD.According to other embodiments, the disease is GA.

[0394] In some embodiments, the method includes selecting a patient with dry AMD. In some embodiments, the method includes selecting a patient with advanced dry AMD. In some embodiments, the method includes selecting a patient with dry AMD with GA. In some embodiments, the method includes selecting a patient with advanced dry AMD with GA. In some embodiments, the method includes selecting a patient with best corrected visual acuity (BCVA) of 20 / 200 or less. In some embodiments, the method includes selecting a patient with best corrected visual acuity (BCVA) of 20 / 63 to 20 / 250. In some embodiments, the method includes selecting a patient with best corrected visual acuity (BCVA) better than 20 / 250. In some embodiments, the method includes selecting a patient with best corrected visual acuity (BCVA) better than 20 / 100. In some embodiments, the method includes selecting a patient with best corrected visual acuity (BCVA) better than 20 / 63. In some embodiments, the method includes selecting a patient with central GA that includes the macular region. In some embodiments, the method includes selecting a patient with central GA without involving the macular region. In some embodiments, the method includes selecting a patient with peripheral GA. In some embodiments, the method includes selecting a patient with central and peripheral GA. In some embodiments, the method includes selecting a patient with a GA size of about 0.2 mm. 2 This includes selecting patients with the above conditions.

[0395] The findings described herein support the unique perspective that RPE cell transplantation according to the teachings of the present invention can replace or rescue retinal cells in patients suffering from retinal pathology or degeneration.Importantly, examples of widespread loss after OpRegen transplantation have been disclosed in the peripheral areas of incomplete RPE and outer retinal atrophy (iRORA), away from primary atrophic lesions (see, for example, Figure 21).

[0396] How to assess clinical improvement In some embodiments, the present disclosure provides a method for assessing the progression of areas of retinal atrophy in the retina following transplantation of retinal pigment epithelial (RPE) cells, comprising the steps of: defining areas of geographic atrophy or complete RPE and outer retinal atrophy (cRORA) within the retinal external limiting membrane (ELM) border; defining the ELM border as the border of atrophy using optical coherence tomography (OCT), where the ELM border is accepted as the delimitation of an area with near-complete histological photoreceptor depletion; marking and measuring the ELM border or ELM descent with OCT at each examination; and determining the area (e.g., mm2) contained within the ELM border at each examination. 2 The method may include calculating the area of ​​atrophy (units) and utilizing a square root transformation (SQRT) of the calculated area to assess change over time relative to the eye itself or relative to a control eye; and defining the progression of atrophy by comparing the calculated area of ​​atrophy between two or more different tests using the OCT and ELM boundaries as boundaries.

[0397] In some embodiments, the method may involve manually measuring and calculating the ELM boundary and the area within the boundary.

[0398] In some embodiments, measurement and calculation of the ELM boundary is performed automatically by the OCT device, by a standalone algorithm, and optionally using artificial intelligence for automatic detection by specific layers, area and volume detection, and prediction of growth.

[0399] In some embodiments, the atrophy can be incomplete RPE and outer retinal atrophy (iRORA) according to the Atrophy Conference Classification (CAM) Study Group Consensus Classification.

[0400] In some embodiments, the change in atrophy area is measured in mm 2 and SQRT.

[0401] In some embodiments, the tests are performed at about 12 months, about 24 months, and about 36 months, respectively.

[0402] In some embodiments, in some aspects, the comparison of the atrophic area uses predicted growth from historical data of the atrophic area and uses a SQRT linear growth calculation to predict the theoretical size of the atrophic area at any future time point.

[0403] In some embodiments, the control group to which the rate of growth of geographic atrophy is compared is a theoretical prediction of growth in the same eye.

[0404] In some embodiments, the comparison of atrophy area is in mm 2 and SQRT may be performed between the treatment and fellow eyes.

[0405] In some embodiments, the comparison of atrophy area is in mm 2 and SQRT may be performed between treated and control eyes.

[0406] In some embodiments, the comparison of areas of atrophy is performed on multiple eyes.

[0407] In some embodiments, the first time point can be before transplantation of RPE cells. In some embodiments, the first time point can be at the time of transplantation of RPE cells. In some embodiments, the first time point can be after transplantation of RPE cells.

[0408] In some embodiments, the time before transplantation can be varied to encompass multiple time points ranging from 1-5 days / weeks / year, hi some embodiments, the time after transplantation can be varied to encompass multiple time points ranging from 1 day to 10 days / weeks / year.

[0409] In some embodiments, the second time point is after the first time point. Thus, the second time point can be anywhere from 1 week to 10 years after the first time point. In some embodiments, the second time point can be anywhere from 1 week to 10 years after transplantation of RPE cells.

[0410] In some embodiments, the present disclosure provides a method for assessing retinal recovery or regeneration in areas within atrophy, using OCT and, optionally, one or more stand-alone algorithms using OCT and optionally artificial intelligence for automated detection of areas and volumes by specific layers and prediction of growth or kinetics. Assessment of recovery or regeneration may be performed by one or more examinations of the retina, the method comprising the steps of defining and using OCT biomarkers as boundaries of any retinal layer; marking and measuring the boundaries of any retinal layer using OCT; calculating the length / width and volume of the specific retinal layer; defining the level of recovery or regeneration by comparing the ELM areas calculated from steps (a)-(c); and detecting newly existing ELM areas.

[0411] In some embodiments, the retinal layer is an ONL and the method detects a newly present ONL region. In some embodiments, the retinal layer is an OPL and the method detects a newly present OPL region. In some embodiments, the retinal layer is an ellipsoid zone and the method detects a newly present ellipsoid zone region. In some embodiments, the retinal layer is a photoreceptor cell and the method detects a newly present photoreceptor cell region. In some embodiments, the retinal layer is an RPE cell layer and the method detects a newly present RPE region.

[0412] In some embodiments, the retinal layers are calculated in combination.

[0413] In some embodiments, OCT examinations are performed at about 12 months, about 24 months, and about 36 months.

[0414] In some embodiments, the comparison of retinal layers is performed on the same eye. In some embodiments, the comparison of areas of atrophy is performed between the treated eye and the fellow eye. In some embodiments, the comparison of areas of atrophy is performed between the treated eye and the control eye. In some embodiments, the comparison of areas of atrophy is performed on multiple eyes.

[0415] In some embodiments, the area of ​​RPE recovery includes when the ELM, ONL and OPL are all present.

[0416] In some embodiments, new areas of RPE are defined by a change in the IR image as new areas of greyish depigmentation.

[0417] In some embodiments, the area of ​​atrophy may be defined by a border or area of ​​preserved ONL.

[0418] In some embodiments, the area of ​​atrophy may be defined by a border or area of ​​preserved OPL.

[0419] In some embodiments, the area of ​​atrophy may be defined by a border or area of ​​preserved RPE.

[0420] In some embodiments, the area of ​​atrophy may be defined by a combination of any or all of the preserved borders or areas of the ONL, ELM, OPL and RPE.

[0421] In some embodiments, the disclosure provides a method for determining areas of geographic atrophy or complete RPE and outer retinal atrophy (cRORA) within the retinal external limiting membrane (ELM) border; determining the ELM border as the border of atrophy using optical coherence tomography (OCT), where ELM border descent is accepted as the demarcation of an area with near-complete histological photoreceptor depletion; marking and measuring the ELM border or ELM descent at each examination using OCT; determining the area (e.g., mm 2 and utilizing a square root transformation (SQRT) of the calculated area to assess change over time relative to the eye itself or relative to a control eye; and defining the progression of atrophy by comparing the calculated atrophy area between two or more different exams using the OCT and ELM boundaries as boundaries.

[0422] In some embodiments, the present disclosure provides methods for assessing clinical improvement by defining and using OCT biomarkers as boundaries of any retinal layer; using OCT to mark and measure boundaries of any retinal layer; calculating the length / width and volume of specific retinal layers; defining the level of recovery or regeneration by comparing ELM areas calculated from steps (a)-(c); and detecting newly existing ELM areas.

[0423] In some embodiments, the clinical improvement is selected from BCVA normal light and low light with or without computer assistance, microperimetry, reading speed, color test with or without computer assistance, flicker test, cone sensitivity and rod sensitivity.

[0424] In some embodiments, the area of ​​retinal atrophy is advanced geographic atrophy, early geographic atrophy, high-risk AMD, or late intermediate AMD.

[0425] Additional material explaining the intended scope of the present invention is submitted herewith as Appendix A, which is incorporated herein by reference in its entirety.

[0426] Device For the methods provided herein, in embodiments the methods include a device or apparatus as described, presented or described herein.

[0427] In one aspect, there is provided a device and / or composition for use in the methods, devices and compositions described, illustrated or exemplified herein.

[0428] In some embodiments, the present disclosure provides a delivery device for use in any of the methods described herein.

[0429] In some embodiments, the device comprises a needle, a capillary and a tip. In some embodiments, the device comprises a needle having an outer diameter of about 0.63 mm and an inner diameter of about 0.53 mm, a capillary having an outer diameter of about 0.5 mm and an inner diameter of about 0.25 mm, and a tip having an outer diameter of about 0.12 mm and an inner diameter of about 0.07 mm.

[0430] In embodiments, the compositions, methods and devices may utilize allogeneic ("off the shelf") product candidates. For example, this may mean that the material is derived from cell lines rather than individual patients, facilitating large scale production and lower production costs than patient-specific treatments.

[0431] The methods, apparatus, compositions, and the like may include those described in the accompanying drawings, which are incorporated herein by reference. EXAMPLES

[0432] The following examples illustrate certain embodiments of the present invention and are not intended to limit the scope of the invention.

[0433] The embodiments of the present specification are further described by the following examples and detailed protocols.However, the examples are intended to merely illustrate the embodiments and should not be interpreted as limiting the scope of the present specification.The contents of all references and published patents and patent applications cited throughout this application are incorporated herein by reference.

[0434] Example 1: Interim Results of a Phase I / IIa Clinical Trial of OpRegen in 24 Patients OpRegen was evaluated in a Phase I / IIa open-label, dose-escalating safety and efficacy study of a single injection of human retinal pigment epithelial cells derived from an established pluripotent cell line and implanted subretinally in advanced dry AMD patients with GA. The study enrolled 24 patients in four cohorts. The first three cohorts enrolled subjects with advanced stages of the disease. All 12 subjects in the first three cohorts were legally blind, had a best-corrected visual acuity (BCVA) of 20 / 200 or less, and had advanced GA (approximately 17 mm in size). 2 The fourth cohort enrolled 12 subjects who presented at an earlier stage of the disease compared with cohorts 1–3 and had better visual acuity (20 / 63–20 / 250 visual acuity) and smaller GA area (up to 11 mm 2 ). Cohort 4 also included subjects treated with OpRegen's new "thaw and inject" (TAI) formulation, which is shipped directly to the site and can be used immediately once thawed, eliminating the complexities and logistics of having to use a dose preparation facility. The first three subjects in Cohort 4 were treated with the previous formulation, and the last nine subjects in Cohort 4 were treated with the "TAI" formulation. The primary objective of this study was to evaluate the safety and tolerability of OpRegen as assessed by the incidence and frequency of treatment-emergent adverse events. Secondary objectives were to evaluate the preliminary efficacy of OpRegen treatment by assessing changes in ophthalmic parameters measured by various methods of primary clinical relevance. Additional objectives included evaluating the safety of delivery of OpRegen using the Gyroscope SDS.

[0435] Twelve subjects treated in cohort 4 had better baseline visual acuity and smaller areas of geographic atrophy (GA). In cohorts 1-3, subjects who were legally blind at baseline experienced visual acuity (VA) decline as expected due to progressive GA. In cohort 4, smaller areas of GA and higher baseline best-corrected visual acuity (BCVA), improved or sustained BCVA, were observed in 11 / 12 (92%) subjects at the last visit (range of -7 to +19 ETDRS letters). OpRegen was well tolerated in all treated subjects (N=24), including two subjects who were less immunosuppressed (COVID or other health conditions). No acute or delayed inflammation and sustained elevated intraocular pressure (IOP) were observed. All subjects reported at least one adverse event (AE), but the majority of AEs were mild (87%). Ocular-related AEs (n=165 events) included: n=136 in pars plana vitrectomy (PPV)-treated subjects (n=17 subjects; 54.7 years F / U) and n=29 in Orbit SDS-treated subjects (n=7 subjects; 6.9 years F / U). Persistent subretinal pigmentation suggested multiyear durability of OpRegen. Anatomical and functional improvements continue to be observed in some subjects, including reduction in drusen, recovery of photoreceptor and RPE layers, localized slowing of GA progression in the treated areas, better visual acuity by ETDRS scores and reading speed, and improved NEI Visual Functioning Questionnaire (VFQ-25) scores (National Eye Institute Visual Functioning Questionnaire-25 (NEI VFQ-25) version 2000 - Interviewer-administered format). Post-treatment surgical intervention occurred in 4 cases (5 events in 4 subjects), including the following: 3 surgically peeled epiretinal membranes (ERMs) (ERMs were observed in 15 of 17 subjects, most were not clinically significant), retinal detachment (RD) was observed in 2 of 17 subjects who received cells via PPV retinotomy, and treatment-responsive choroidal neovascularization (CNV) was observed in 3 Orbit SDS-treated subjects, all of whom received a single dose of an approved anti-VEGF drug.OpRegen TAI formulation was administered to seven Orbit SDS-treated and two PPV-treated subjects. Slow resorption of subretinal fluid was observed in four Orbit SDS / TAI-treated subjects without sequelae. Evaluation of clinical benefit is ongoing and utilizes detailed OCT analysis in addition to standard FAF measurements. Long-term follow-up of subjects is ongoing.

[0436] As part of an ongoing attempt to administer the minimum effective dose and duration of immunosuppressive therapy, immunosuppression was only utilized for approximately 3 months of perioperative time in cohort 4 subjects. Of note, one OpRegen patient who received a modified immunosuppressive regimen at baseline that did not include tacrolimus and included only mycophenolate mofetil, showed no signs of acute or delayed inflammation or rejection of OpRegen cells at 4.5 months post-transplant. One patient was diagnosed with COVID immediately after the procedure and all immunosuppression was stopped and resumed once the patient became asymptomatic. This second patient similarly showed no signs of acute or delayed inflammation or rejection of OpRegen cells at 4.5 months post-op. Other than the reduced regimen described above, immunosuppressants were discontinued as scheduled, typically within 90 days post-op, and no cases of acute or delayed rejection or inflammation due to OpRegen were reported.

[0437] Nine subjects were treated with OpRegen's new "thaw and inject" (TAI) formulation and seven were treated using the Gyroscope Orbit™ Subretinal Delivery System (Orbit SDS). Representative FP images of pigmented areas in the GA of treated eyes are shown 3 months (Figure 1) and 9 months (Figure 2) after treatment. The pigmented areas are evidence of the presence of RPE cells in the GA.

[0438] Overall, 11 / 12 (92%) of treated eyes of Cohort 4 subjects had baseline visual acuity or better at 4.5 months to more than 3 years after implantation. Improvements in best-corrected visual acuity (BCVA) reached a maximum of +19 letters on the Early Treatment Diabetic Retinopathy Study (ETDRS) chart. In contrast, 11 / 12 (92%) of the subjects' untreated eyes were below baseline entry values ​​at the same time point. Among the newly reported data, three (50%) of the recently treated Cohort 4 subjects showed notable improvements in BCVA ranging from +7 to +16 letters at the last scheduled evaluation at least 4.5 months. Two additional Cohort 4 subjects experienced a 2-letter gain from baseline values. One patient was 7 letters below baseline. The previously reported retinal structural improvement and reduction in drusen density in some subjects continues. Evidence of durable engraftment of OpRegen RPE cells extended to more than 5 years in the earliest treated subjects. The trend for slower progression of GA in treated compared with fellow eyes continued. Overall, OpRegen was well tolerated, with no unexpected or serious adverse events.

[0439] The data in Tables 1, 2 and 3 below summarize the change in scores for five subjects (14, 15, 13, 16 and 17) in Cohort 4. All five subjects saw improvement in the vision category. The average change in scores for all five subjects combined in the vision category was 18%.

[0440] The subject in Cohort 4, who had evidence of retinal recovery and a history of confirmed GA growth (first reported at 9 months), maintained a smaller area of ​​GA than at baseline at month 23. This subject also experienced further improvement in BCVA 9-23 months post-treatment, whereas the untreated eye experienced further decline in visual acuity.

[0441] Individual changes in visual acuity over time (from 1 month to 24 months) for Cohort 4 are shown in Figure 3 (measured by change in ETDRS letters from baseline) and Figure 8 (measured by reading speed). The mean change in visual acuity (measured by change in ETDRS letters from baseline) is shown in Figure 5. The mean change in GA size in treated eyes is shown in Figure 4.

[0442] OpRegen cells, as described herein, are a suspension of allogeneic RPE cells that have the potential to counteract RPE cell loss in GA by supporting retinal structure and function. Study eligibility criteria, dosing, and objectives are shown in Table 13 below. Perioperative immunosuppressive therapy included tacrolimus 0.01 mg / kg administered daily for up to 6 weeks after surgery, and mycophenolate up to 2.0 g administered daily for at least 3 months after surgery.

[0443] Table 13: Design criteria, dosing and objectives of Phase I / IIa study. Subretinal delivery was via vitrectomy / retinotomy (n=17), with suprachoroidal cannula using Orbit SDS® (Gyroscope Therapeutics) used in cohort 4 only (n=7). TIFF2024522608000002.tif50170

[0444] Individual subject data are shown in Figure 6 and Figures 7A-7C.

[0445] (Table 1) Mean total change across all subjects and categories TIFF2024522608000003.tif243137

[0446] Table 2. Mean total change across all subjects and categories TIFF2024522608000004.tif24229

[0447] (Table 3) This table shows how many of the 5 subjects in Cohort 4 showed improvement in each category. TIFF2024522608000005.tif24169

[0448] The blank questionnaire containing all questions (National Eye Institute Visual Functioning Questionnaire 25 (VFQ-25) Version 2000 - Interviewer-Administered Form) is incorporated herein by reference. Questionnaires were administered at Screening Visit 11, Visit 17, Visit 18, Visit 19, Visit 20, Visit 21 and Visit 22. The items shown in Table 4 were averaged to generate the VFQ-25 subscales.

[0449] (Table 4) TIFF2024522608000006.tif93170

[0450] Observations from clinical trial data include improved quality of life, improved reading speed, and improved microperimetry.

[0451] Example 2: Retinal Recovery in Subjects with Dry AMD with GA Retinal recovery is difficult to observe because the cells used herein do not autofluoresce under FAF, a common imaging technique used to measure GA borders. Measurement by IR has not been accepted as a method to assess atrophy borders. High-resolution OCT is an alternative to FAF for measuring GA lesion borders and fine layers of the retina. Using OCT in this way is a slower manual process with its own limitations, but offers the ability to distinguish individual cell types within the retina, like layers of a cake (e.g. ONL, OPL, RPE). Figures 9, 12-14, 16, 18-22, 26-28, and 30 show several cross-sectional and "air-to-air" views of the atrophic areas at baseline and after treatment.

[0452] Subject 14 had anatomical improvement of OPL, ONL, ELM, RPE and outer retina regeneration / recovery at 9 and 23 months post-treatment (Figures 9-15). Similarly, subject 21 showed a reduction in the GA border at 1 month, anatomical improvement and recovery of the ELM (Figures 16 and 17), as well as near complete recovery of the previously atrophic area (separated from the temporary GA), with regeneration of the defect layer and "disappearance" of the atrophic lesion (Figure 18). Improvement was seen at 2 and 3 months post-treatment (Figures 18-22). RPE delivery to the GA was observed in subject 14 during the treatment procedure as well as at 2 and 3 months post-treatment (Figure 31).

[0453] Microperimetry. Figure 15 shows preliminary evidence that the recovered areas may also be functional (simply seeing tissue does not mean the tissue is active). Microperimetry involves flashing a pinpoint light onto the retina to "map" the area used for vision. Microperimetry data are difficult to collect, so they only exist for a few subjects at a few time points. However, they provide at least some evidence that the patient 14 has visual capabilities in the recovered areas.

[0454] Subject 22 showed improvement in visual acuity and GA size in treated eyes compared to untreated eyes (Figure 23). Pigmentation at 3 months post-treatment in subject 22 indicated the presence of RPE cells (Figure 24). GA size as measured by IR imaging demonstrated a decrease in the GA border at 3 months (Figure 25), as did OCT measurements (Figures 26-30).

[0455] Subject 14 was followed for 35 months. A separate tissue layer was detectable at 23 months, but absent at 9 months. There were numerous examples of this phenomenon throughout the observation period and throughout the entire (peripheral) area of ​​atrophy. Applicants measured the patient's GA growth rate in the year prior to treatment, allowing for extrapolation of the patient's GA size based on untreated growth rate. GA remained unchanged compared to baseline for 3 years, which was unexpected given the natural history of the disease (i.e., the situation gets progressively worse). The patient's treated eye only recently fell below baseline, but remains much better than the contralateral eye, which the patient no longer uses for vision. Subject 14 is the original case and shows persistence of effect.

[0456] New findings in subject 21. A similar observation was detected as early as 2.5 months in another patient. Analysis was performed on the outer retinal region only. Baseline showed expected GA / cRORA with loss of ELM, EZ in expected locations. After 3 weeks, significant outer retinal changes were observed including apparent partial remodeling of the ELM / EZ. Diffuse thickening of the EZ and amorphous hyperreflective subretinal material was present. At 6 weeks, some EZ changes persisted, but loss of the EZ also occurred. RPE / Bruch thickening was also observed.

[0457] New findings in subject 22. Subject 22 is a woman who called her treatment experience "life changing." New material and expansion of the ELM in various locations around the GA was identified, as well as several small areas or "islands" of GA that were not connected to the main areas. By 3 months, these islands had disappeared after treatment, supporting the contention that early intervention results in better clinical outcomes in dry AMD. Patient 22 was treated with Orbit SDS.

[0458] Baseline showed central GA / cRORA with multifocal satellites. Expected loss of EZ / ELM / hyperpermeance was observed through the RPE. At 4 weeks, there was macular hole formation with large subretinal fluid collection. Numerous deposits on the RPE surface were identified with IR and OCT. At 6 weeks, residual subretinal fluid and new material was evident on the surface of the RPE. PED was evident with highly hyperreflective inner material (possible type 1 CNV). By 3 months, all subretinal fluid had resolved, with residual subretinal material and large central subretinal deposits appearing. There was new epiretinal inner fluid. By 4 months, elongation of the ELM was noted in many places. Subretinal material increased. Retinal hemorrhage on fundus photography corresponded to areas with fluid, possible type 1 CNV through Bruch. FAF showed overall expansion of RPE loss, but increased pigmentation and ELM extension to the border of defined atrophy.

[0459] Abstract In subjects 14, 21 and 22, cases in which transplanted cells recovered comprised the majority of GAs. Cell placement appears to be important to achieve these results, with important implications for Orbit evaluation. After seeing recovery in subject 14 (patient with complete coverage of the GA), surgeons made greater efforts to deliver cells across the GA in the final seven subjects. In the final four Orbit subjects, only one successfully deposited cells across the GA, despite being in the hands of a highly trained surgeon. In contrast, both of the procedures that accessed PPV were able to achieve this successfully (PPV is more flexible in this regard). In the third case (patient no. 22), partial coverage was achieved using Orbit by the same surgeon who completed the complete coverage.

[0460] Although recovery did not perfectly correlate with clinical outcome at this time, some interesting connections can be drawn, but given that recovery has not been observed previously with any other approach to treating AMD, there is no precedent to help predict the dynamics of functional regeneration, if functional recovery occurs.

[0461] Example 3: Key Control Endpoints for Dry and Wet Forms of Age-Related Macular Degeneration (AMD) The anticipated efficacy endpoints are: Primary Efficacy Endpoint Total area (mm2) of GA lesion(s) in the study eye based on FAF 2 ) from baseline to month 12.

[0462] Key secondary efficacy endpoints: 1) Change from baseline in monocular reading speed (study eye) as assessed by Minnesota Reading (MNRead) or Radner Reading Charts at Month 24 (selected countries). 2) Change from baseline in Functional Reading Independence Index (FRII) composite score at Month 24. 3) Change from baseline in normal luminance best corrected visual acuity score (NL-BCVA) at Month 24 as assessed by ETDRS charts. 4) Change from baseline in low luminance best corrected visual acuity score (LL-BCVA) at Months 12 and 24 as assessed by ETDRS charts. 5) Change from baseline in low luminance deficiency (LLD) at Months 12 and 24. 6) Total area (mm2) of GA lesion(s) in study eye as assessed by FAF (selected sites). 2 ) Change from baseline at each scheduled assessment. 7) Change from baseline in monocular limiting print size (study eye) at 12 and 24 months (selected countries) as assessed by MNRead or Radner Reading Charts. 8) Change from baseline in National Eye Institute Visual Functioning Questionnaire 25 item version (NEI VFQ-25) distance activity subscale score at 12 and 24 months. 9) Number of scotomas assessed by mesopic microperimetry for evaluation of macular functional response (Oaks study only). 10) Change in macular sensitivity assessed by mesopic microperimetry for evaluation of macular functional response. 11) Systemic plasma concentrations of APL-2 over time.

[0463] Safety endpoints: 1) incidence and severity of ocular and systemic treatment-emergent adverse events, 2) incidence of anti-treatment antibodies against APL-2, and 3) incidence of new active CNV in the study eye.

[0464] Details on some of the key secondary endpoints in the dry AMD trial include:

[0465] Change from baseline in the number of absolute scotomas assessed by mesopic microperimetry at week 48 [Time Frame: Baseline, Week 48]. Scotoma are test points of microperimetry that are centered on the macula and report the lack of retinal sensitivity within the range tested; a maximum of 68 points were tested within this range. Higher results indicate enlargement of the absolute scotoma and a higher number of absolute scotoma. Mesopic microperimetry evaluation was performed after dilation in the test eye only, and data were transferred to a central reading center. Data were collected up to week 48 instead of week 96 due to early termination of the study. A positive change from baseline indicates an increase in the number of absolute scotoma (more lack of retinal sensitivity); worsening of the disease.

[0466] Change from baseline in mean macular sensitivity as assessed by mesopic microperimetry at week 48 [Time frame: baseline, week 48]. Macular sensitivity was assessed using mesopic microperimetry, and evaluations were performed after dilation in the study eye only, and data were transferred to a central reading center. A negative change from baseline indicates a decrease in mean macular sensitivity; disease worsening. Data were collected through week 48 instead of week 96 due to early termination of the study.

[0467] Change from baseline in best corrected visual acuity (BCVA) score as assessed by the Early Treatment Diabetic Retinopathy Study (ETDRS) chart at week 48 [Time Frame: Baseline, Week 48]. BCVA score was based on the number of letters correctly read on the ETDRS visual acuity chart assessed at a starting distance of 4 meters (m). BCVA score test was performed before dilating the eye. BCVA score ranges from 0 to 100 letters in the test eye. The fewer letters correctly read on the eye chart, the worse the vision (or visual acuity). A negative change from baseline indicates poorer visual acuity; worsening of the disease. Data were collected through week 48 instead of week 96 due to early termination of the study.

[0468] Percentage of participants with a loss of <15 letters from baseline in BCVA score at week 48 [Time Frame: Week 48]. Loss of <15 letters from baseline was assessed by ETDRS chart at a starting distance of 4 meters (m). BCVA was measured using an eye chart and reported as the number of letters correctly read (range 0-100 letters). The fewer letters correctly read on the eye chart, the worse the vision (or visual acuity). Data were collected through week 48 instead of week 96 due to early termination of the study.

[0469] Change from baseline in low-light visual acuity (LLVA) as assessed by the ETDRS chart under low-luminance conditions at week 48 [Time frame: baseline, week 48]. LLVA was measured by having participants read a normally illuminated ETDRS chart with a 2.0-log unit neutral density filter over the best correction in that eye. Assessments were performed before dilating the eye. LLVA scores range from 0 to 100 letters in the test eye. The fewer letters correctly read on the eye chart, the worse the vision (or visual acuity). Data were collected through week 48 instead of week 96 due to early termination of the study.

[0470] Percentage of participants with less than 15 letter loss from baseline in LLVA score at week 48 [Time frame: week 48]. Loss of less than 15 letters from baseline was assessed by ETDRS chart at a starting distance of 4m. Data were collected through week 48 instead of week 96 due to early termination of the study.

[0471] Change from baseline in binocular reading speed assessed by the Minnesota Low Vision Reading Test (MNRead) chart or Radner reading chart at 48 weeks [Time Frame: Baseline, Week 48]. The MNRead acuity card was a continuous text reading acuity card suitable for measuring reading acuity and reading speed in normal and low vision participants. The MNRead acuity card consisted of a single simple sentence with an equal number of letters. A stopwatch was used to record the time to the tenth of a second. Sentences that could not be read or were not attempted due to vision should be recorded as time 0 and errors 10. The Radner Reading Card was suitable for measuring reading speed, reading acuity, and marginal print size. The reading test was stopped when the reading time exceeded 20 seconds or when the participant made significant errors. A negative change from baseline indicates a decrease in binocular reading speed; a worsening of the disease. Data were collected until week 48 instead of week 96 due to early termination of the study.

[0472] Change from baseline in maximum monocular reading speed assessed by MNRead chart or Radner reading chart at week 48 [Time frame: baseline, week 48]. MNRead acuity cards were continuous text reading acuity cards suitable for measuring reading acuity and reading speed in normal and low vision participants. MNRead acuity cards consisted of a single simple sentence with an equal number of letters. A stopwatch was used to record the time to the tenth of a second. Sentences that could not be read or were not attempted due to vision should be recorded as time 0 and errors as 10. Radner Reading Cards were suitable for measuring reading speed, reading acuity, and marginal print size. Reading trials were stopped when the reading time exceeded 20 seconds or when the participant made significant errors. A negative change from baseline indicates a decrease in monocular reading speed; a worsening of the disease. Data were collected up to week 48 instead of week 96 due to early termination of the study.

[0473] Change from baseline in the National Eye Institute Visual Functioning Questionnaire 25-item (NEI VFQ-25) version composite score at week 48 [time frame: baseline, week 48]. The NEI-VFQ-25 questionnaire contains 25 items based on which an overall composite VFQ score and 12 subscales were derived: near activities, distance activities, general health, overall visual acuity, eye pain, vision-specific social functioning, vision-specific mental health, vision-specific role difficulties, vision-specific dependency, driving, color vision and peripheral vision. Responses to each question were converted to a 0–100 score. For each subscale, total score = mean of items contributing to the score. For each subscale and total score, score range: 0–100, higher scores represent better functioning. A negative change from baseline indicates poorer visual function; worsening disease. Data were collected up to week 48 instead of week 96 due to early termination of the study.

[0474] Change from baseline in NEI VFQ-25 near activity subscale score at week 48 [Time frame: baseline, weeks]. The NEI-VFQ-25 questionnaire contains 25 items based on which near activity was measured. Near activity is defined as reading plain print in a newspaper, performing a job or hobby that requires near vision, or finding something on a crowded shelf. Responses to each question were converted to a 0–100 score. Subscale = average of items contributing to the score. In this subscale, the score range is 0–100, with higher scores representing better functioning. A negative change from baseline indicates a decrease in near visual activity; disease worsening. Data were collected up to week 48 instead of week 96 due to the early termination of the study.

[0475] Change from baseline in NEI VFQ-25 distance activity subscale score at week 48 [Time frame: baseline, week 48]. The NEI-VFQ-25 questionnaire contains 25 items based on which distance activity was measured. Distance activity is defined as reading a street sign or store name and descending stairs, steps, or curbs. Responses to each question were converted to a 0–100 score. Subscale = average of items contributing to the score. In this subscale, the score range is 0–100, with higher scores representing better function. A negative change from baseline indicates a decrease in distance visual activity; disease worsening. Data were collected up to week 48 instead of week 96 due to early termination of the study.

[0476] Change from baseline in mean Functional Reading Independence (FRI) index at week 48 [Time frame: baseline, week 48]. The FRI was an interviewer-administered questionnaire with 7 items regarding the functional reading activities most relevant to GA AMD participants. It has one overall index score. For each FRI index reading activity performed in the past 7 days, participants were asked about the extent to which they required visual aids, coordination of the activity, or assistance from another participant. The mean FRI index score ranged from 1 to 4, with higher scores indicating greater independence. A negative change from baseline indicates a decline in FRI; disease worsening. Data were collected through week 48 instead of week 96 due to early termination of the study.

[0477] Example 4: SD-OCT imaging to measure thickness and area The thickness, area and volume of different layers of the retina were determined in treated eyes. SD-OCT images were captured using a Spectralis (Spectralis; Heidelberg Engineering, Inc., Heidelberg, Germany), and the macular volume consisted of 512 × 49 equally spaced B-scans within a 20 × 20 degree field of view centered on the fovea. Retinal layers of all B-scans were manually segmented for thickness and area measurements using 3D-OCTOR (developed at Doheny Eye Institute). Specifically, the outer nuclear layer, photoreceptor inner segments (myoid zone), photoreceptor outer segments (ellipsoid zone), and RPE + drusen complex were manually segmented using all B-scans within the macular volume.

[0478] Exemplary B-scans are shown in Figures 33A-C. The AB scan (Figure 33A) was divided into layers based on the boundaries (Figure 33B) and the layer thicknesses and areas were determined (Figure 33C). Thickness maps show the thickness of the whole retina, ONL, photoreceptor outer segments, RPE+drusen complex (Figure 34, left to right respectively), and photoreceptor inner segments. Exemplary thickness maps for individual subjects are shown in Figures 35-52. Results are shown in Tables 5-10.

[0479] Table 5. SD-OCT parameters of total macular volume at baseline and 6 months in study eyes TIFF2024522608000007.tif23590

[0480] Table 6. SD-OCT parameters of total macular volume at baseline and 6 months in fellow eyes TIFF2024522608000008.tif23791

[0481] Table 7. SD-OCT parameters of total macular volume at baseline and 12 months in study eyes TIFF2024522608000009.tif24194

[0482] Table 8. SD-OCT parameters of total macular volume at baseline and 12 months in fellow eyes TIFF2024522608000010.tif24191

[0483] Table 9. SD-OCT parameters of total macular volume at baseline and 6 months in study eyes - Cohort 4 TIFF2024522608000011.tif240103

[0484] Table 10. SD-OCT parameters of total macular volume at baseline and 6 months in fellow eyes - Cohort 4 TIFF2024522608000012.tif239102

[0485] Table 11. SD-OCT parameters of total macular volume at baseline and 12 months in study eyes - Cohort 4 TIFF2024522608000013.tif23994

[0486] Table 12. SD-OCT parameters of total macular volume at baseline and 12 months in fellow eyes - Cohort 4 TIFF2024522608000014.tif24299

[0487] Example 5: RPE treatment results in restoration of the blood-retinal barrier. RPE secretes very high levels of PEDF (measured for OpRegen levels of 2000-4000ng / ml / day), which contributes to the therapeutic efficacy. PEDF is a 50kDa protein secreted by RPE and Müller glia in vivo, with beneficial functions such as antiangiogenic activity, neuroprotective function for photoreceptors by restoring mitochondrial dynamics disturbed by aging and oxidative stress, anti-inflammatory activity (by interacting with master factor NF-KappaB), and anti-fibrotic activity by binding to extracellular matrix (collagen and proteoglycans). In OpRegen-treated subjects, this is evidenced by improved fluorescein angiography (FA) in patients with and without drusen, and OCT imaging with possible signs of ECM remodeling or scar attenuation within GA lesions as early as 2-4 weeks after implantation.

[0488] Baseline FA examination in subject 8 showed a large amount of fluorescein dye leaking into the vitreous cavity, thereby blocking the visibility of vascular perfusion during the choroidal flush and arterial phase, suggesting that blood-retinal barrier disruption and parainflammation were pre-existing in the eye (Figure 53). At 22 months post-implantation, FA examination showed clear choroidal and retinal vascular perfusion with no dye leaking into the vitreous cavity, indicating that OpRegen restored the integrity of the disrupted BRB, likely through multiple mechanisms of action (e.g., via various targets of PEDF). Figures 54A-D provide three additional examples of BRB restoration or repair by OpRegen cell therapy.

[0489] Subject 8 is a typical example of a patient with extensive drusen that spread throughout the posterior retina. Figure 55 shows that drusen clearance began in the superior graft area (upper left) and moved inferiorly to clear almost the entire posterior area, except for a small elongated band that remained 8 months postoperatively (top, second from left, large circle). OCT imaging features were consistent with color fundus photography at 5.5 months (top, second from right) and 8 months (bottom, second from right) compared to baseline (upper right and lower right); subRPE drusen were significantly reduced or disappeared. The host retinal tissue appears attenuated, suggesting possible ECM remodeling, in part and possibly due to the biological effects brought about by the presence of high levels of PEDF.

[0490] At 11 months, in subject 8, the graft continued to remodel the host retina after the large drusen had disappeared (Figures 56A-C). FA showed significantly reduced staining (drusen), but appeared to have a veil-like membrane obscuring the retinal vasculature. At 22 months on fundus examination, the retinal tissue appears clearer compared to that at baseline, possibly due to its anti-inflammatory effect, or ECM cleansing, where PEDF has a role in regulating extramatrix turnover.

[0491] Figure 57 provides a time course of FA examination from early, mid and late stages, demonstrating significant improvement in retinal health, better visibility of vascular perfusion throughout, and reduced inflammation. The retinal tissue appears very clean; this FA pattern has not been reported previously with other treatment modalities. This is highly unique to the therapeutic effect of OpRegen.

[0492] All references provided herein (including all non-patent literature, patents, and patent publications) are hereby incorporated by reference in their entirety.

[0493] Example 6: Clinical Trial Design Utilizing External Limiting Membrane (ELM) Biomarkers to Assess Dry AMD ELM biomarker clinical trial objectives 1. To demonstrate that OpRegen cells slow the progression of atrophic areas (the current gold standard for clinical trials in atrophic AMD). 2. To demonstrate that OpRegen cells restore / regenerate the outer retina in areas within atrophy (a completely novel paradigm not addressed by current approaches for atrophic AMD) 3. Demonstrate at least some trend toward improved visual function, as required by the Food and Drug Administration (FDA) and other regulatory agencies. 4. The methodology, selected biomarkers, and clinical trial design will be selected to maximize prior points and be pre-specified from those learned in the ongoing OpRegen Phase 1 / 2a clinical trial (the "ongoing trial"), as described in Example 1.

[0494] Methods and Biomarkers Used in Phase 1 / 2a Clinical Trial in Example 1 Background: In recent years, fundus autofluorescence (FAF) has been used as an anatomical biomarker to measure the area of ​​atrophy and assess its progression over time [1-3].

[0495] It is currently believed that measuring atrophic lesions by FAF in ongoing clinical trials involving allografted RPE is not sufficient to probe their full functionality, as they were too early in their lifespan to have had enough time to accumulate enough lipofuscin to become autofluorescent. Furthermore, FAF does not take into account the state of the retinal layers, and therefore cannot provide an indication of potential retinal repair.

[0496] Measurement using infrared imaging (IR) alone is not accepted for assessing the atrophy border.

[0497] There has been a trend towards optical coherence tomography (OCT)-based measurements of atrophy in the retina field, due to improved scanning resolution, layer detection, and advances in expert knowledge.[4-7] Measurement of the atrophy border can be extremely accurate without the potential artifacts and limitations of FAF, but allows for a simultaneous and accurate assessment of the status of all retinal layers[8], especially the RPE, ellipsoid zone (EZ), external limiting membrane (ELM), outer nuclear layer (ONL), and outer plexiform layer (OPL).

[0498] Using the presence of a new layer of hyperreflectivity on Bruch's membrane in areas of geographic atrophy or cRORA (complete RPE and outer retinal atrophy) as a biomarker for engrafted OpRegen RPE cells may compromise accuracy as it is currently not possible to prove that these are actually viable cells and not just cell debris or non-viable cells using current technology.

[0499] Therefore, to consider that the highly reflective new layer at the location of the RPE is in fact a newly transplanted functional RPE, signs of outer retinal recovery such as a new EZ, a new ELM, a new ONL, and new or less subsidence of the OPL must be present.

[0500] Imaging Biomarkers: Primary: ELM border. The descending ELM border is recognized as the delimitation of an area with near-total photoreceptor depletion and therefore as the border of atrophy in cRORA [9].

[0501] In ongoing trials, the ELM border or ELM descent was measured manually on every scan to define the border of atrophy. However, the exact ELM border is questionable due to the resolution / quality of the scan, which may reduce accuracy. Despite this limitation, overall, the results are consistent despite imperfect accuracy. Potential optical scanning misalignments were taken into account. The results are consistent, indicating that there may be some potential small misalignments that go undetected.

[0502] Secondary: ONL and OPL. Measurement of new areas of ONL and OPL have been used as support and proof of the concept of retinal recovery / regeneration. However, despite the visibility of new ONL, on many images even new, less subsided OPL was visible. For consistency, the border of atrophy was based only on the ELM, even if the ELM was in a less favorable position than the recovery suggested by the new ONL and OPL. This approach was considered the most conservative.

[0503] RPE: To avoid misinterpretations that may be due to non-functional cells or cellular debris, new areas of RPE repair with OpRegen cells or new RPE layers were considered only if ELM, ONL and OPL were present.

[0504] Designing future clinical trials The biomarkers and endpoints selected will ultimately influence the design of future trials and also dictate the sample size. A randomized trial would seem ideal, but has many challenges. The large variability of atrophic progression in this patient population due to the very large phenotypic heterogeneity would suggest that a very large number of patients would be used. However, even if patients were stratified by at least three different phenotypes (GA associated with mainly soft drusen, GA associated with reticular drusen, and GA associated with mixed drusen), enrollment, completion, and interpretation would be difficult.

[0505] With other clinical trials for GA available, or drugs that may be approved within the next 2 years, it would be difficult to maintain GA patients in the control group long-term without treatment. The only ethical solution for these patients would probably be to allow crossover to the treatment group after a predefined period of 12 months.

[0506] Despite the randomization of patients, the surgical nature of the study makes it impossible to mask patients and therefore offers little advantage over using the fellow eye as a study control. Due to the limited number of patients showing signs of recovery in the current study, it may be too early to move to a larger Phase 2b / 3 trial.

[0507] Nonrandomized trials using better defined, prespecified biomarkers and endpoints, better entry VA, and better baseline and historical information, and comparing the fellow eye, would potentially be more desirable.

[0508] Study Design: A non-randomized controlled trial using the study eye itself, and the fellow eye. The advantages of comparing the fellow eye are numerous. There is no need to compare with a phenotypically matched control eye, as the phenotype is the same. It eliminates the need to maintain a control eye for as long as 3 years, which would be unethical in control patients and would preclude treatment with other trials or drugs that may be approved later as they become available. The nature of the surgical intervention makes masking of study patients difficult, if not impossible, for study site personnel. However, optometrists and imaging graders could potentially be masked.

[0509] Anatomical endpoints: Some endpoints will focus on change in lesion size (the current gold standard in clinical trials) and retinal regeneration / recovery (a complete new treatment efficacy paradigm).

[0510] Primary endpoint: (Change in lesion size in mm by SQRT). The primary endpoint will be a comparison of the actual lesion size growth and the predicted growth at a specific time point (probably 12 months) according to the historical progression of the study eye itself.

[0511] Lesion size is measured in mm using square root transformation (SQRT). Comparison of atrophy areas is performed in mm 2and SQRT are performed between treated and fellow eyes of the same patient, avoiding the problem of comparing eyes with different atrophy sizes.

[0512] If very small and very large lesions are excluded, the growth rate of the lesion is considered and accepted to be linear as measured by SQRT. Thus, historical growth could predict the growth rate of the lesion at any time if the eye was untreated.

[0513] Additionally, the magnitude of potential error due to measurement variability using the SQRT has been reported.[8] The SQRT of lesion area measurements appears to eliminate the confounding variable of baseline lesion size and the need to stratify lesions based on baseline lesion size. Thus, the study eye and fellow eye could be compared regardless of whether the lesions differed in size.

[0514] Therefore, the primary endpoint was the change in lesion size measured by SQRT, as measured by OCT using the edge of the ELM, compared with the predicted growth of the study eye at 12 months (secondary endpoints at 24 and 36 months).

[0515] To accurately predict growth, good and reliable historical data of at least a 6-month period is required. Optionally, if no historical data is available, patients may be enrolled in the trial 6 months prior to transplant. Accurate historical data is important for the success of the trial and accurate prediction of regression growth over time. If this information is accurate, changes in atrophic growth velocity will be detectable and reliable.

[0516] Figure 11 shows the theoretical growth of the study eye and fellow eye of patient 120 of the ongoing clinical trial (dotted line). The study eye had a much smaller actual growth at M33 than the predicted growth. In fact, the size of the lesion after 3 years was the same compared to baseline. The actual growth of the fellow eye was slightly smaller than the theoretical growth, but it was very close, differing only by 0.1 mm per year. This supports the hypothesis that historical data must be as accurate as possible and must be derived from reliable and consistent imaging. Moreover, it must be taken into account that some degree of error should be considered and factored into the study design. [8]

[0517] Anatomical secondary endpoints to assess change in lesion size:Secondary endpoints are change in lesion size measured by OCT using the edge of the ELM compared to growth in the fellow eye, measured by SQRT.Change in lesion size measured by OCT using the edge of the ELM compared to predicted growth in the study eye after 24 and 36 months, measured by SQRT.

[0518] Anatomical secondary endpoints to evaluate retinal regeneration / recovery: Change in lesion size measured by SQRT measured by OCT using the edge of the ELM compared to BASELINE size at 12, 24 and 36 months (Figure 62). For retinal repair, this endpoint results in a negative number, meaning that the lesion size was reduced.

[0519] Figure 63 shows the change in area of ​​viable RPE at 12, 24 and 36 months within / outside the boundary of the lesion at baseline (defined as the area with a highly reflective monolayer overlying Bruch's membrane, with ELM, ONL and PLEXIFORM above it). Figure 64 shows the change in volume of ONL at 12, 24 and 36 months within / outside the boundary of the lesion at baseline (ONL defined when OPL and / or ELM are present). Automated detection and artificial intelligence will be employed for this endpoint in particular.

[0520] Visual Function Secondary Endpoints: BCVA and LLVA: Relatively preserved visual function at baseline shall be mandatory to be in a position to detect differences between the study and comparison eyes at 12, 24 or 36 months. Therefore, the entry criterion for BCVA at baseline shall be approximately 20 / 40. The study eye shall be the worst eye in order not to penalize BCVA and LLVA results. Ideally, both eyes shall have similar BCVA with less than 10 letters difference in order to be consistently comparable.

[0521] Secondary endpoints for visual acuity: • Change in BCVA and LLVA at 12, 24 and 36 months compared with baseline. • Change in BCVA and LLVA at 12, 24 and 36 months compared with the fellow eye.

[0522] Microperimetry: To be in a position to detect differences between the study eye and the comparison eye (fellow eye) at 12, 24 or 36 months, the ability at baseline to perform microperimetry is mandatory for both eyes and lack of ability shall be an exclusion criterion.

[0523] Key secondary endpoints: • Change in microperimetry at 12, 24 and 36 months compared with baseline. • Changes in microperimetry at 12, 24 and 36 months compared with the fellow eye.

[0524] (Patient-reported outcome measure): If the test eye is the patient's best eye, this is not a good visual acuity endpoint.

[0525] FRI-Functional Reading Independence (FRI) Index

[0526] NEI VFQ-25 - National Eye Institute Vision Function Questionnaire; see, e.g., www.nei.nih.gov / sites / default / files / 2019-06 / vfq_sa.pdf, incorporated herein by reference in its entirety.

[0527] Abstract OBJECTIVE: To evaluate efficacy, defined as reduction in lesion growth rate and retinal repair / regeneration, as well as safety and tolerability, in patients with geographic atrophy secondary to AMD treated with transplantation of OpRegen cells.

[0528] phase. Phase 2b

[0529] Study population and key inclusion criteria: ● Patients with bilateral AMD and geographic atrophy and no history of wet AMD in either eye. (As an alternative option in case of difficulty in bilateral recruitment, a second group of patients with unilateral GA without exudation could be considered. These eyes could not be compared with their fellow eyes, but with the fellow eyes of the bilateral group. In this group, only half of the secondary endpoints would apply. In this option, the number of treated eyes would be twice as many as the control eyes, 2:1). ● BCVA was better than 20 / 80 in both eyes. ● The difference between the two eyes is less than 10 letters. ● To justify intervention despite a relatively good BCVA at baseline, but at the same time, eyes with at least partial cavity atrophy (approximately 50%) and no cavity atrophy but very good visual acuity are excluded, since they would likely guarantee a decline in function in the fellow eye without treatment. • Ability to perform binocular microperimetry.

[0530] Study Design: ● Single-masked non-randomized study Comparator / control group: Predicted growth of GA and changes in outer retinal characteristics in the same eye ○ GA growth in the other eye Functional endpoints compared to baseline in the same eye Functional endpoints compared with the fellow eye

[0531] Endpoints Primary endpoint. Change in lesion size measured by SQRT, measured by OCT using the edge of the ELM, and compared with predicted growth in the study eye after 12 months.

[0532] Secondary endpoints:

[0533] Anatomical examination. Changes in lesion size measured by SQRT as measured by OCT using the edge of the ELM and compared with predicted growth in the study eye at 24 and 36 months.

[0534] Changes in lesion size measured by SQRT and by OCT using the edge of the ELM compared with growth in the fellow eye.

[0535] Changes in lesion size measured by SQRT, measured by OCT using the edges of the ELM, and compared with baseline growth after 12, 24, and 36 months.

[0536] Change in area of ​​viable RPE at 12, 24 and 36 months within / outside the lesion border at baseline.

[0537] Change in volume of viable ONL at 12, 24 and 36 months within / outside the lesion margin at baseline.

[0538] Function: Change in BCVA at 12, 24 and 36 months compared with baseline.

[0539] Change in LLVA at 12, 24 and 36 months compared with baseline.

[0540] Change in BCVA at 12, 24, and 36 months compared with the fellow eye.

[0541] Change in LLVA at 12, 24, and 36 months compared with the fellow eye.

[0542] Change in microperimetry at 12, 24 and 36 months compared with baseline.

[0543] Changes in microperimetry at 12, 24 and 36 months compared with the fellow eye.

[0544] (Quality of life: only if the best eye is treated, vision endpoint is unlikely, not best case scenario).

[0545] Changes from baseline to 12, 24, and 36 months in the FRI study.

[0546] Change from baseline to 12, 24, and 36 months on the NEI VFQ-25 study.

[0547] Example 7: Additional Data from a Phase 1 / IIa Study of the Safety and Activity of OpRegen in Geographic Atrophy (GA) Secondary to Age-Related Macular Degeneration (AMD) As mentioned above, the Phase I / IIa study was an open-label, single-arm, multicenter, dose-escalation study evaluating a single dose of OpRegen delivered subretinal in patients with bilateral GA. Twenty-four patients were enrolled in four cohorts. The first three cohorts enrolled only legally blind patients with best-corrected visual acuity (BCVA) of 20 / 200 or better. The fourth cohort enrolled 12 patients with visual impairment (smaller mean area of ​​GA and BCVA of 20 / 65 to 20 / 250). Cohort 4 also included patients treated with a new "thaw and inject" formulation of OpRegen, which is shipped directly to the site and can be used immediately once thawed, eliminating the complexities and logistics of having to use a dose preparation facility. The primary objective of the study was to evaluate the safety and tolerability of OpRegen as assessed by the incidence and frequency of treatment-emergent adverse events. A secondary objective is to evaluate the preliminary activity of OpRegen treatment by assessing changes in ophthalmic parameters measured by various methods of primary clinical relevance.

[0548] Baseline characteristics and follow-up are shown below in Table 14. Greater disease severity was observed in cohorts 1-3 vs. cohort 4.

[0549] Table 14. Baseline characteristics and study follow-up TIFF2024522608000015.tif65170 a The worse eye based on BCVA was selected for subretinal delivery of OpRegen. b It is based on central grading of fundus autofluorescence imaging.

[0550] A safety summary was provided, indicating that OpRegen was well tolerated with an acceptable safety profile. All 24 (100%) patients treated reported ≥1 AE and ≥2 ocular AEs (the most frequent systemic AE was URTI (n=7), the most frequent ocular AEs were conjunctival hemorrhage / hypemia (n=17) and ERM (n=16)). The majority of reported AEs (87% in cohorts 1–3; 93% in cohort 4) were mild. No clusters of AEs related to immunosuppressive therapy were reported. One patient discontinued due to an AE (stage IV lung adenocarcinoma unrelated to treatment). No cases of rejection following subretinal delivery of OpREgen were reported. No acute or delayed intraocular inflammation or persistent intraocular pressure elevation was observed.

[0551] Ocular AEs with OpRegen are reported below in Table 15.

[0552] (Table 15) Ocular AEs with OpRegen were primarily related to the surgical procedure for subretinal delivery. TIFF2024522608000016.tif76170 a 7 / 12 (58%) patients in cohorts 1-3 and 5 / 12 (42%) in cohort 4 had ERM at baseline; 610 patients with reported ERMAEs in cohorts 1-3 and 2 / 6 in cohort 4 had pre-existing ERM. b Clinical significance indicates ERM requiring surgical intervention.

[0553] Subretinal delivery of OpRegen to the GA region and fovea demonstrated greater visual functional gains along with areas of improvement in outer retinal structures. Five patients in Cohort 4 had OpREgen delivered to most or all of the GA region, including the fovea. These five patients had greater improvements in visual function (mean gain of 12.8 letters) with evidence of clearly improved areas of outer retinal structures as assessed by SD-OCT (Figure 69).

[0554] The study further included evaluation of GA after OpRegen delivery, showing the advantage of SD-OCT over fundus autofluorescence (FAF) imaging (Figure 70). The allogeneic hESC-derived RPE cells in OpRegen are young and have low lipofuscin content, and therefore it was expected that OpRegen RPE cells would not be easily detected by standard FAF after subretinal delivery. Greater hyperreflectivity was seen in the RPE / Bruch's membrane (Figure 71). SD-OCT imaging suggested the presence of OpRegen in areas of previous GA. SD-OCT demonstrated improvement of the outer retinal structure when OpREgen was delivered to the GA area. As shown in Figure 72, focal disruption of the RPE layer, choroidal hyperpermeability, and outer retinal subsidence at baseline were no longer present at 12 months, and scan alignment was confirmed by the presence of prominent drusen and choroidal vascular markings. Furthermore, as shown in Figures 73A and 73B, at 12 months compared to baseline, cRORA features were no longer present, there was greater hyperreflectivity at the level of the RPE / Bruch's membrane, less choroidal hypertransmission, retinal subsidence was eliminated, and there was greater continuity of the outer retina, with similar features at the nasal, superior, and inferior borders of the GA in this case. Figure 74 showed improvement of the outer retina near the fovea.

[0555] Twelve-month primary endpoint data from this Phase I / IIa study suggested that OpRegen was well tolerated and demonstrated a safety profile with mostly mild AEs. Ocular AEs observed with OpRegen were primarily related to the surgical procedure used for subretinal delivery. Preliminary evidence of improvement in extraretinal structure and visual function with OpRegen was observed in patients with GA and visual impairment (cohort 4; n=12). All 24 treated patients reported at least one adverse event (AE) and at least one ocular AE. The majority of AEs reported with OpRegen were mild (cohorts 1–3, 87%; cohort 4, 93%), and immunosuppressive therapy was well tolerated. Ocular AEs observed with OpRegen were primarily related to the surgical procedure used for subretinal delivery, the most common of which were conjunctival hemorrhage / hyperemia (n=17) and epiretinal membrane (n=16). One patient discontinued the study due to an AE unrelated to the procedure. No cases of rejection, acute or delayed intraocular inflammation, or sustained increases in intraocular pressure were reported after subretinal delivery of OpRegen. Preliminary evidence of improved visual function was observed in patients with GA and visual impairment at baseline (Cohort 4 [n=12]). Cohort 4 patients gained a mean of 7.6 letters of visual acuity at 12 months in the study eye. Three patients (25%) in Cohort 4 gained 15 or more letters of visual acuity at 12 months in the study eye. Five patients in Cohort 4 in whom OpRegen was delivered to most or all of the GA area, including the fovea, had greater increases in visual function (mean gain of 12.8 letters) and evidence of areas of clear improvement in extraretinal structures as assessed by SD-OCT. These data support the potential for OpRegen to slow, halt, or reverse disease progression in GA. To confirm these findings, the optimal stage for intervention, the surgical procedure for subretinal delivery, and the targeted delivery site of OpRegen require further evaluation in larger, controlled clinical trials.

[0556] These data support the potential for OpRegen to slow, halt, or reverse disease progression in GA.

[0557] References: TIFF2024522608000017.tif132155

Claims

1. A method for evaluating the progression of retinal atrophy regions in the retina of a subject after transplantation of retinal pigment epithelial (RPE) cells, comprising: a) at a first time point, defining an area of geographic atrophy or complete RPE and outer retinal atrophy (cRORA) within the outer limiting membrane (ELM) boundary of the retina; b) using optical coherence tomography (OCT) to mark and measure the ELM boundary or ELM boundary decline, wherein the ELM boundary is the boundary of atrophy and the ELM boundary decline is a definition of a region where photoreceptor cells are histologically almost completely depleted, the step of marking and measuring the ELM boundary or ELM boundary decline; c) calculating the area contained inside the ELM boundary to define a first calculated area and determining the square root transformation (Sqrt) of the first calculated area; and d) defining the degree of progression of the atrophy by comparing the Sqrt of the first calculated area with a control A method comprising the steps of:

2. The method according to claim 1, further comprising repeating steps a) to c) at a second time point to determine the Sqrt of a second calculated area, wherein the control is the Sqrt of the second calculated area.

3. The method according to claim 1, wherein the control is the past degree of progression of the retina.

4. The method according to claim 1, wherein the control is the degree of progression of a control retina.

5. The method according to claim 4, wherein the control retina is the untreated retina of the subject.

6. The method according to claim 1, wherein the measurement of the ELM boundary and the calculation of the first calculated area are performed manually.

7. The method according to claim 1, wherein the measurement of the ELM boundary is automatically performed by the OCT device using a stand-alone algorithm.

8. The measurement and calculation of the ELM boundary are performed using artificial intelligence for automatic detection, detection of area and volume by specific layers, and prediction of growth The method according to claim 7.

9. The method according to claim 1, wherein the atrophy is incomplete RPE and outer retinal atrophy (iRORA) according to the Consensus Classification of the Atrophy Meeting (CAM) Research Group.

10. The progression of the retinal atrophy area, measured in both mm ２ and SQRT, according to the method of claim 1.

11. The method according to claim 2, wherein steps a) to c) are performed at a third time point.

12. The method according to claim 11, wherein the first time point, the second time point, and the third time point are about 12 months, about 24 months, and about 36 months after transplantation, respectively.

13. The method according to claim 3, wherein the past rate of progression is growth predicted from past data of the atrophic region, and the theoretical size of the atrophic region at any future time point is predicted using SQRT line growth calculation.

14. The method according to any one of claims 1 to 13, wherein the control group for comparing the growth rate of geographic atrophy is the theoretical prediction of growth in the same eye.

15. The comparison of the atrophy regions is in mm ２ The method according to any one of claims 1 to 13, carried out between the treated eye of the subject and the other eye using both AND SQRT.

16. The calculation is in mm ２ The method according to any one of claims 1 to 13, wherein the calculation is performed in

17. The method according to any one of claims 1 to 13, wherein the comparison of the atrophic region is performed on a plurality of eyes.

18. The method according to any one of claims 1 to 13, wherein the first time point is before transplantation of RPE cells.

19. The method according to any one of claims 1 to 13, wherein the first time point is at the time of transplantation of RPE cells.

20. The method according to any one of claims 1 to 13, wherein the first time point is after transplantation of RPE cells.

21. A method for evaluating the restoration or regeneration of the retina in a region within the atrophic region, comprising: a) defining and using an OCT biomarker as the boundary of any retinal layer; b) using OCT to mark and measure the boundary of any retinal layer; c) calculating the length / width and volume of a specific retinal layer; d) defining the level of restoration or regeneration by comparing the ELM regions calculated from steps (a) to (c); and e) detecting newly existing ELM regions The method comprising.

22. The method according to claim 21, wherein the retinal layer is the ONL, and the method detects newly existing ONL regions.

23. The method according to claim 21, wherein the retinal layer is the OPL, and the method detects newly existing OPL regions.

24. The method according to claim 21, wherein the retinal layer is the ellipsoid zone, and the method detects newly existing ellipsoid zone regions.

25. The method according to claim 21, wherein the retinal layer is photoreceptor cells, and the method detects newly existing photoreceptor cell regions.

26. The method according to claim 21, wherein the retinal layer is the RPE cell layer, and the method detects newly existing RPE regions.

27. The method according to any one of claims 21 to 26, wherein the retinal layers are calculated in combination.

28. The method according to any one of claims 21 to 26, wherein the OCT examination is performed at about 12 months, about 24 months, and about 36 months.

29. The method according to any one of claims 21 to 26, wherein the comparison of the retinal layers is performed on the same eye.

30. The method according to any one of claims 21 to 26, wherein the comparison of the atrophic regions is performed between the treated eye and the other eye.

31. The method according to any one of claims 21 to 26, wherein the comparison of the atrophic regions is performed between the treated eye and the control eye.

32. The method according to any one of claims 21 to 26, wherein the comparison of the atrophic regions is performed on a plurality of eyes.

33. The method according to any one of claims 21 to 26, wherein the region of RPE recovery is a case where all of ELM, ONL, and OPL are present.

34. A method for evaluating the clinical improvement according to claim 1, wherein the clinical improvement is selected from the group consisting of BCVA normal light and low light with or without computer assistance, microperimetry, reading speed, color test with or without computer assistance, flicker test, cone sensitivity, and rod sensitivity.

35. A method for evaluating the clinical improvement according to claim 21, wherein the clinical improvement is selected from the group consisting of BCVA normal light and low light with or without computer assistance, microperimetry, reading speed, color test with or without computer assistance, flicker test, cone sensitivity, and rod sensitivity.

36. The method according to any one of claims 1 to 13, 21 to 26, 34 to 35, wherein the retinal atrophic region is advanced geographic atrophy, early geographic atrophy, high-risk AMD, or late intermediate AMD.

Images