Compositions and methods for restoring or preventing loss of vision caused by disease or traumatic injury

Abstract

To provide: a method for one or more of, treating retinal damage, slowing the progression of retinal damage, preventing retinal damage, replacing retinal tissue and restoring damaged retinal tissue; and a retinal tissue graft.SOLUTION: Bioprosthetic retinal grafts (or devices) comprising stem cell derived tissues and / or cells may be used to slow the progression of retinal degenerative disease, slow the progression of retinal degenerative disease after traumatic injury, slow the progression of age related macular degeneration (AMD), prevent retinal degenerative disease, prevent retinal degenerative disease after traumatic injury, prevent AMD, restore retinal pigment epithelium (RPE), photoreceptor cells (PRCs) and retinal ganglion cells (RGCs) lost from disease, injury or genetic abnormalities, in a subject, or for other purposes. Bioprosthetic retinal grafts may comprise a bioprosthetic carrier or scaffold suitable for implantation into the ocular space of a subject's eye, to form a bioprosthetic retinal patch.SELECTED DRAWING: None

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to and the benefit of U.S. Provisional Patent Application No. 62 / 539,542, filed July 31, 2017, U.S. Provisional Patent Application No. 62 / 577,154, filed October 25, 2017, U.S. Provisional Patent Application No. 62 / 593,228, filed November 30, 2017, U.S. Provisional Patent Application No. 62 / 646,354, filed March 21, 2018, and U.S. Provisional Patent Application No. 62 / 665,483, filed March 1, 2018, each of which is incorporated herein by reference in its entirety. [Background technology]

[0002] background Retinal degeneration (RD) diseases ultimately result in the degeneration of photoreceptors (PR) and are the third leading cause of blindness worldwide. Genetic conditions, age, and trauma (military and civilian) are the main causes of retinal degeneration-related vision loss. Once the photoreceptors have degenerated, current technology is incapable of repairing the retina and restoring vision.

[0003] In developed countries, age-related macular degeneration (AMD) is the leading cause of RD in people over the age of 55. Approximately 15 million people in the United States currently suffer from AMD, accounting for approximately 50% of all vision loss in the United States and Canada. Retinitis pigmentosa (RP) is the most common cause of inherited visual impairment, with a prevalence of 1:4000, affecting an estimated 50,000-100,000 people in the United States and approximately 15 million people worldwide. Other retinal diseases that result in severe vision loss include Leber's congenital amaurosis (LCA), a rare genetic disorder in which vision loss is often caused by a congenital retinal insufficiency. The degree of vision loss varies from patient to patient but can be quite severe (near to no light perception).

[0004] While personal protective equipment to protect the head and torso during combat has improved, soldiers are increasingly sustaining injuries to less protected body parts, such as the face and eyes. Eye injuries from blast exposure are the fourth most common injury sustained in military combat. Eye injuries often lead to blindness, resulting in significant loss of quality of life and independence. While penetrating injuries often result in severe tissue damage or loss, non-penetrating or closed eye injuries can similarly result in destruction of the higher tissue architecture of the eye, causing retinal detachment, photoreceptor cell death, and optic nerve destruction, resulting in irreversible vision loss. Closed eye injuries often exhibit an injury pattern in which the ocular structures remain largely intact, but intervention is required to prevent retinal and optic nerve degeneration resulting in devastating vision loss.

[0005] A recently developed strategy to restore vision in RD patients is the implantation of electronic neuroprosthetic chips, which introduce light-capturing sensors into the subretinal space to electrically transmit visual signals to the remaining neurons in the patient's retina. One problem with this approach is that ongoing retinal degeneration, retinal remodeling and thinning, and gliosis can gradually separate the electronic components from the body, reducing the interaction between the chip and the retina that is essential for transducing electrical signals. An additional problem arises in living tissue, where the metals and wiring used in the chips can oxidize due to body fluids, limiting the stability of electronic devices in living tissue.

[0006] Retinal tissue transplants using human fetal retinas have also been demonstrated to restore visual perception in blind animals and improve vision in patients with retinal degeneration. Although this approach is promising, generating a new layer of healthy human retina in the patient's subretinal space, the use of fetal tissue as a therapeutic option is hindered by ethical issues and because of the scarce and unpredictable supply of fetal tissue. Furthermore, the success of this vision restoration procedure relies on the selection of a human fetal retina of a specific developmental age (8-17 weeks) and its precise placement in the patient's subretinal space. Adult human retinas are generally not suitable for this application, as they die soon after transplantation.

[0007] Retinal stem cell therapy stands out as one of the most pressing unsolved problems among all stem cell replacement therapies. The eye is a small, immune-privileged, enclosed organ. It is possible to access the intraocular space for transplantation, and the retina can be visualized by non-invasive methods. However, repairing the neural retina by replacing functional cells is a complex task. For best results, the new cells need to migrate to specific locations in the retinal layers and re-establish specific synaptic connectivity with the host. Synaptic remodeling of neural circuits in advanced RD further complicates this task.

[0008] Thus, there is a need for robust and feasible treatments for vision restoration technologies targeted at restoring and protecting structure and function following retinal injury or disease that may result in severe retinal destruction, or that may affect large portions of the retina, or that may result in progressive degeneration over time.

[0009] The present disclosure addresses these and other shortcomings in the field of regenerative medicine and cell therapy. Summary of the Invention

[0010] overview In one aspect, a method is provided for one or more of treating retinal damage, slowing the progression of retinal damage, preventing retinal damage, replacing retinal tissue, and repairing damaged retinal tissue, the method comprising applying an hESC-derived retinal tissue graft to a subject.

[0011] In another aspect, methods are provided for one or more of slowing the progression of a retinal degenerative disease, slowing the progression of a retinal degenerative disease after traumatic injury, slowing the progression of age-related macular degeneration (AMD), slowing the progression of an inherited retinal disease, stabilizing a retinal disease, preventing a retinal degenerative disease, preventing a retinal degenerative disease after traumatic injury, improving vision or visual perception, preventing AMD, restoring retinal pigment epithelium (RPE), photoreceptor cells (PRCs), and retinal ganglion cells (RGCs) lost due to disease, injury, or genetic abnormality, increasing RPE, PRCs, and RCGs, or treating RPE, PRC, and RCG deficiencies, comprising applying an hESC-derived retinal tissue graft to a subject.

[0012] In another aspect, the retinal damage is caused by one or more of blast exposure, genetic disorders, retinal diseases, and retinal damage. In another aspect, the retinal disease includes retinal degenerative diseases. In another aspect, the retinal damage is caused by one or more of age-related macular degeneration (AMD), retinitis pigmentosa (RP), and Leber's congenital amaurosis (LCA).

[0013] In one embodiment, a method is described for using hESC-derived retinal tissue, comprising retinal pigment epithelial (RPE) cells, retinal ganglion cells (RGC) and photoreceptor (PR) cells. In another embodiment, the RPE cells, RGC cells and PR cells are arranged such that there is a central layer of retinal pigment epithelial (RPE) cells, and radially outward from the RPE cell layer there is a retinal ganglion cell (RGC) layer, a layer of secondary retinal neurons (corresponding to the inner nuclear layer of mature retina), a photoreceptor (PR) cell layer and an outer layer of RPE cells. In another embodiment, each of the layers comprises differentiated cells that are characteristic of cells in the corresponding human retinal tissue layer. In another embodiment, each of the layers comprises progenitor cells, and after application, some or all of the progenitor cells differentiate into the mature cells of the corresponding human retinal tissue layer.

[0014] In another embodiment, each layer comprises substantially fully differentiated cells. In yet another embodiment, the hESC-derived retinal tissue further comprises a biocompatible scaffold for forming a bioprosthetic retinal patch. In another embodiment, the bioprosthetic retinal graft comprises about 10,000 to 100,000 photoreceptor cells. In another embodiment, several pieces of hESC-derived retinal tissue are added to the biocompatible scaffold to form a large bioprosthetic patch. In another embodiment, the hESC-derived retinal tissue graft, or dissociated cells of the hESC-derived retinal tissue graft, can deliver one or more of neurotrophic factors, neurotrophic exosomes, and mitogens to a subject. In yet other embodiments, the neurotrophic factors and mitogens include one or more of brain-derived neurotrophic factor (BDNF), glial-derived neurotrophic factor (GDNF), neurotrophin-34 (NT34), neurotrophin 4 / 5, nerve growth factor-beta (βNGF), proNGF, PEDF, CNTF, the pro-survival mitogen basic fibroblast growth factor (bFGF=FGF-2), and pro-survival members of the WNT family.

[0015] In another aspect, application of the hESC-derived retinal tissue graft preserves the thickness of the retinal layer at the application site for about 1 to about 3 months. In yet another aspect, the application further comprises administration of an immunosuppressant. In another aspect, the application comprises the use of epinephrine before, during, and / or after application of the retinal graft.

[0016] In yet other aspects, an immunosuppressant is administered before, during, and / or after application.

[0017] In other embodiments, the method further comprises modulating intraocular pressure. In other aspects, modulating intraocular pressure occurs before, during, and / or after application of the retinal tissue.

[0018] In certain aspects, the tissue is applied using an intraocular implantation tool.

[0019] In other embodiments, the hESC-derived retinal tissue is applied sub- or epi-retinal.

[0020] In other embodiments, the application of hESC-derived retinal tissue grafts results in tumor-free integration of the hESC-derived retinal tissue with the subject's retinal tissue.

[0021] In other embodiments, integration of the retinal graft occurs about 2-10 weeks after application. In other embodiments, integration includes structural integration. In other embodiments, integration includes functional integration and occurs about 1-6 months after application. In other embodiments, application does not cause retinal inflammation.

[0022] In other embodiments, after application, the retinal tissue undergoes lamination.

[0023] In another embodiment, after administration, retinal tissue neurons are + , K + and / or Ca ++ Shows signs of current.

[0024] In other embodiments, the method further comprises demonstrating connectivity between the retinal tissue and the pre-existing tissue, hi other embodiments, the binding is demonstrated by one or more of WGA-HRP transsynaptic tracer, histology, IHC, or electrophysiology.

[0025] In other aspects, the method further comprises measuring the level of functional recovery.

[0026] In other embodiments, the level of functional recovery comprises an increase in electrophysiological response of at least 10% over baseline.

[0027] In another aspect, a retinal tissue graft is provided for transplantation into a subject's eye, the retinal tissue graft comprising retinal pigment epithelial (RPE) cells, retinal ganglion cells (RGCs), second order retinal neurons, and photoreceptor (PR) cells, wherein the RPE cells, RGC cells, and PR cells are configured to form a central core.

[0028] In other embodiments, there are between about 1,000 and 250,000 photoreceptors.

[0029] In other aspects, the second order retinal neurons correspond to the inner nuclear layer of the mature retina.

[0030] In other embodiments, the cells are arranged radially outward from the core, and the retinal tissue includes a retinal ganglion cell (RGC) layer, a layer of secondary retinal neurons, a layer of photoreceptor (PR) cells, and an outer layer of RPE cells. In other embodiments, the graft includes between 1,000 and about 250,000 cells.

[0031] In other embodiments, the graft is implanted in the subretinal or epiretinal space.

[0032] In other embodiments, the graft is implanted into the subretinal or epiretinal space near the macula, hi other embodiments, increased synapse formation occurs concomitantly with increased electrical activity.

[0033] In other embodiments, after transplantation, the neurons integrate the graft with existing tissue.

[0034] In other embodiments, the neurons are CALB2 positive. In other embodiments, connectivity is demonstrated by WGA-HRP transsynaptic tracer. In other embodiments, after transplantation, axons connect the graft to existing tissue. In other embodiments, the axons are CALB2 positive.

[0035] In other embodiments, after transplantation, the cells of the graft mature into RGCs.

[0036] In other embodiments, after transplantation, the graft forms synapses with existing neurons.

[0037] In other embodiments, after implantation, the graft forms a bond with the existing tissue.

[0038] In another embodiment, the bond forms between 1 day and about 5 weeks after implantation.

[0039] In other embodiments, after implantation, the graft forms axons that span the pre-existing tissue ONL.

[0040] In another embodiment, the graft produces paracrine factors.

[0041] In other embodiments, the paracrine factors are produced before and / or after application.

[0042] In other embodiments, the graft produces a neurotrophic factor.

[0043] In other embodiments, the graft produces neurotrophic factors either before or after application.

[0044] In other embodiments, the neurotrophic factor comprises one or more of BDNS, GDNF, bNGF, NT4, bFGF, NT34, NT4 / 5, CNTF, PEDF, a serpin, or a WNT family member.

[0045] In another embodiment, after transplantation, the level of functional recovery is measured as an increase in electrophysiological response.

[0046] In other embodiments, the level of functional recovery is measured as an increase in electrophysiological response to at least 10% of baseline.

[0047] In other embodiments, after implantation, the axons of the graft penetrate and integrate into existing tissue.

[0048] In other aspects, the tissue is derived from human pluripotent stem cells.

[0049] In other aspects, the grafts are useful for slowing the progression of a retinal degenerative disease, slowing the progression of a retinal degenerative disease after traumatic injury, slowing the progression of age-related macular degeneration (AMD), slowing the progression of an inherited retinal disease, stabilizing a retinal disease, preventing a retinal degenerative disease, preventing a retinal degenerative disease after traumatic injury, improving vision or visual perception, preventing AMD, restoring retinal pigment epithelium (RPE), photoreceptor cells (PRCs), and retinal ganglion cells (RGCs) lost due to disease, injury, or genetic abnormality, increasing RPE, PRCs, and RCGs, or treating RPE, PRC, and RCG deficiencies in a subject.

[0050] In other aspects, the graft is capable of tumor-free survival for at least about 6-24 months after implantation into the recipient's intraocular space, with layering and development of the PR and RPE layers, including PR outer segment elongation, synapse formation, electrophysiological activity, and connectivity with the recipient's retinal cells.

[0051] In other embodiments, the graft is capable of extending axons into and integrating into the recipient's outer nuclear layer (ONL), inner nuclear layer (INL), and ganglion cell layer (GCL) 5 weeks after implantation into the intraocular space of the recipient's eye.

[0052] Provided herein are methods for restoring vision loss or slowing the progression of vision loss by application of a retinal patch. In one aspect, a product for restoring or improving vision is provided, which can be injected or introduced into the epiretinal or subretinal space of a patient's eye.

[0053] In another aspect, a method for correcting vision loss in a subject having a damaged retina is provided, the method comprising restoring retinal tissue to the site of the damage. In yet another aspect, a method for correcting vision loss in a subject is provided, the method comprising restoring the damaged retinal tissue by applying a biological retinal patch to the site of the damage. In another aspect, a method for correcting vision loss in a subject having a damaged retina is provided by applying a biological retinal patch, the biological retinal patch comprising engineered retinal tissue; an electrospun biopolymer scaffold; and an adhesive, the retinal tissue being secured to the biopolymer by the adhesive.

[0054] [The present invention 1001] A method for one or more of treating retinal damage, slowing the progression of retinal damage, preventing retinal damage, replacing retinal tissue, and repairing damaged retinal tissue, comprising applying an hESC-derived retinal tissue graft to a subject. [The present invention 1002] 1. A method for one or more of slowing the progression of a retinal degenerative disease, slowing the progression of a retinal degenerative disease after traumatic injury, slowing the progression of age-related macular degeneration (AMD), slowing the progression of an inherited retinal disease, stabilizing a retinal disease, preventing a retinal degenerative disease, preventing a retinal degenerative disease after traumatic injury, improving vision or visual perception, preventing AMD, restoring retinal pigment epithelium (RPE), photoreceptor cells (PRCs), and retinal ganglion cells (RGCs) lost due to disease, injury, or genetic abnormality, increasing RPE, PRCs, and RCGs, or treating RPE, PRC, and RCG deficiencies, comprising applying an hESC-derived retinal tissue graft to a subject. [The present invention 1003] The method of claim 1001, wherein the retinal damage is caused by one or more of blast exposure, a genetic disorder, a retinal disease, and a retinal injury. [The present invention 1004] The method of the present invention 1003, wherein the retinal disease comprises a retinal degenerative disease. [The present invention 1005] The method of claim 1001, wherein the retinal damage is caused by one or more of age-related macular degeneration (AMD), retinitis pigmentosa (RP), and Leber's congenital amaurosis (LCA). [The present invention 1006] The method of any one of claims 1001 to 1002, wherein said hESC-derived retinal tissue comprises retinal pigment epithelial (RPE) cells, retinal ganglion cells (RGC), and photoreceptor (PR) cells. [The present invention 1007] The method of the present invention 1006, wherein the RPE cells, the RGC cells, and the PR cells are configured such that there is a central layer of RPE cells and radially outward from the RPE cell layer there is a retinal ganglion cell (RGC) layer, a layer of secondary retinal neurons (corresponding to the inner nuclear layer of the mature retina), a photoreceptor (PR) cell layer, and an outer layer of RPE cells. [The present invention 1008] The method of claim 1007, wherein each of said layers comprises differentiated cells characteristic of cells in a corresponding layer of human retinal tissue. [The present invention 1009] 1007. The method of claim 1007, wherein each of said layers comprises progenitor cells, and after application, some or all of said progenitor cells differentiate into mature cells of the corresponding human retinal tissue layer. [The present invention 1010] The method of claim 1007, wherein said layer comprises substantially fully differentiated cells. [The present invention 1011] The method of any one of claims 1001 to 1002, wherein said hESC-derived retinal tissue further comprises a biocompatible scaffold for forming a bioprosthetic retinal patch. [The present invention 1012] 1007. The method of claim 10, wherein said bioprosthetic retinal graft comprises about 10,000 to 100,000 photoreceptor cells. [The present invention 1013] The method of claim 1011, wherein several of said hESC-derived retinal tissue fragments are attached to said biocompatible scaffold to form a large bioprosthetic patch. [The present invention 1014] The method of claim 1006, wherein the hESC-derived retinal tissue graft, or dissociated cells of the hESC-derived retinal tissue graft, are capable of delivering one or more of neurotrophic factors, neurotrophic exosomes, and mitogens to a subject. [The present invention 1015] The method of the present invention 1014, wherein said neurotrophic factors and said mitogens comprise one or more of brain-derived neurotrophic factor (BDNF), glial-derived neurotrophic factor (GDNF), neurotrophin-34 (NT34), neurotrophin 4 / 5, nerve growth factor-beta (βNGF), proNGF, PEDF, CNTF, the pro-survival mitogen basic fibroblast growth factor (bFGF=FGF-2), and pro-survival members of the WNT family. [The present invention 1016] The method of invention 1001 or 1002, wherein application of said hESC-derived retinal tissue graft preserves the thickness of the retinal layer at the application site for about 1 to about 3 months. [The present invention 1017] The method of any one of claims 1001 to 1002, further comprising administration of an immunosuppressant. [The present invention 1018] The method of any one of claims 1001 to 1002, further comprising administering epinephrine before, during and / or after application of said retinal graft. [The present invention 1019] The method of claim 1017, wherein said immunosuppressant is administered before, during and / or after said administering. [The present invention 1020] The method of claim 1001, further comprising regulating intraocular pressure. [The present invention 1021] The method of claim 1020, wherein said adjustment of intraocular pressure occurs before, during, and / or after application of said retinal tissue. [The present invention 1022] The method of claim 1001, wherein the tissue is applied using an intraocular implantation tool. [The present invention 1023] The method of any one of claims 1001 to 1002, wherein said hESC-derived retinal tissue is applied subretinal or epiretinal. [The present invention 1024] The method of claim 1001 or 1002, wherein application of said hESC-derived retinal tissue graft results in tumor-free integration of said hESC-derived retinal tissue with said subject's retinal tissue. [The present invention 1025] The method of the present invention, wherein integration of the retinal graft occurs approximately 2 to 10 weeks after application. [The present invention 1026] The method of the present invention 1025, wherein the integration includes structural integration. [The present invention 1027] The method of claim 1024, wherein the integration includes structural integration and occurs about 1 to 6 months after application. [The present invention 1028] The method of claim 1001, wherein the application does not cause retinal inflammation. [The present invention 1029] The retinal tissue graft of the present invention 1026, wherein after application, the retinal tissue undergoes lamination. [The present invention 1030] After application, the retinal tissue neurons + , K + and / or Ca ++The method of the present invention 1001, showing an indication of a current. [The present invention 1031] The method of claim 1001, further comprising verifying connectivity between the retinal tissue and existing tissue. [The present invention 1032] The method of claim 1031, wherein said binding is demonstrated by one or more of WGA-HRP transsynaptic tracer, histology, IHC, or electrophysiology. [The present invention 1033] The method of claim 1001, further comprising measuring the level of functional recovery. [The present invention 1034] The method of the present invention 1033, wherein the level of functional recovery comprises an increase in electrophysiological response of at least 10% over baseline. [The present invention 1035] 1. A retinal tissue graft for transplantation into an eye of a subject, comprising: A retinal tissue graft comprising retinal pigment epithelial (RPE) cells, retinal ganglion cells (RGCs), second order retinal neurons, and photoreceptor (PR) cells, wherein the RPE cells, the RGC cells, and the PR cells are configured to form a central core. [The present invention 1036] 1035. A retinal tissue graft of the present invention having approximately 1,000 to 250,000 photoreceptors. [The present invention 1037] The retinal tissue graft of the present invention 1035, wherein said secondary retinal neurons correspond to the inner nuclear layer of the mature retina. [The present invention 1038] The retinal tissue graft of the present invention 1035, wherein the cells are arranged radially outward from the core, and the retinal tissue comprises a retinal ganglion cell (RGC) layer, a layer of secondary retinal neurons, a layer of photoreceptor (PR) cells, and an outer layer of RPE cells. [The present invention 1039] 1035. A retinal tissue graft of the present invention comprising 1,000 to about 250,000 cells. [The present invention 1040] The retinal tissue graft of the present invention 1035, which is implanted into the subretinal space or epiretinal space. [The present invention 1041] The retinal tissue graft of the present invention 1040 is implanted into the subretinal or epiretinal space near the macula. [The present invention 1042] The retinal tissue graft of the present invention 1035, wherein increased synapse formation occurs concomitantly with increased electrical activity. [The present invention 1043] The retinal tissue graft of the present invention 1035, wherein after transplantation, neurons connect the graft to existing tissue. [The present invention 1044] The retinal tissue graft of the present invention 1043, wherein said neurons are CALB2 positive. [The present invention 1045] Retinal tissue of the present invention 1043, where binding is demonstrated by WGA-HRP transsynaptic tracer. [The present invention 1046] The retinal tissue graft of the present invention 1035, wherein after transplantation, axons connect the graft to existing tissue. [The present invention 1047] The retinal tissue of the present invention 1046, wherein the axons are CALB2 positive. [The present invention 1048] The retinal tissue graft of the present invention 1035, wherein after transplantation, cells of the graft mature into RGCs. [The present invention 1049] The retinal tissue graft of the present invention 1035 forms synapses with existing neurons after transplantation. [The present invention 1050] The retinal tissue graft of the present invention 1035 forms a bond with existing tissue after transplantation. [The present invention 1051] The retinal tissue of the present invention, wherein the bond is formed 1 day to about 5 weeks after transplantation. [The present invention 1052] The retinal tissue graft of the present invention 1035, which after transplantation, forms axons that span the existing ONL tissue. [The present invention 1053] A retinal tissue graft of the present invention 1035, which produces paracrine factors. [The present invention 1054] The retinal tissue graft of the present invention 1053, wherein said paracrine factors are produced before and / or after application. [The present invention 1055] 1035. A retinal tissue graft of the present invention which produces neurotrophic factors. [The present invention 1056] The retinal tissue graft of the present invention 1055, which produces neurotrophic factors before or after application. [The present invention 1057] 1055. The retinal tissue of the present invention, wherein the neurotrophic factor comprises one or more of BDNS, GDNF, bNGF, NT4, bFGF, NT34, NT4 / 5, CNTF, PEDF, a serpin, or a WNT family member. [The present invention 1058] After transplantation, the level of functional recovery is measured as an increase in electrophysiological response, 1035 of the retinal tissue graft of the present invention. [The present invention 1059] The retinal tissue graft of the present invention 1058, wherein the level of functional recovery is measured as an increase in electrophysiological response to at least 10% of baseline. [The present invention 1060] The retinal tissue graft of the present invention, wherein after transplantation, the axons of the graft penetrate and integrate into existing tissue. [The present invention 1061] The retinal tissue graft of the present invention 1035, wherein said tissue is derived from human pluripotent stem cells. [The present invention 1062] The retinal tissue graft of the present invention 1035 is useful for slowing the progression of retinal degenerative disease, slowing the progression of retinal degenerative disease after traumatic injury, slowing the progression of age-related macular degeneration (AMD), slowing the progression of inherited retinal disease, stabilizing retinal disease, preventing retinal degenerative disease, preventing retinal degenerative disease after traumatic injury, improving vision or visual perception, preventing AMD, restoring retinal pigment epithelium (RPE), photoreceptor cells (PRC), and retinal ganglion cells (RGC) lost due to disease, injury, or genetic abnormality, increasing RPE, PRC, and RCG, or treating RPE, PRC, and RCG deficiency in a subject. [The present invention 1063] The retinal tissue graft of the present invention 1035, after implantation into the intraocular space of a recipient, is capable of laminating and developing the PR and RPE layers, and of surviving without tumors for at least about 6 to 24 months, including PR outer segment elongation, synapse formation, electrophysiological activity, and connectivity with the recipient's retinal cells. [The present invention 1064] A retinal tissue graft of the present invention 1035, which is capable of extending axons into the outer nuclear layer (ONL), inner nuclear layer (INL), and ganglion cell layer (GCL) of the recipient five weeks after implantation into the intraocular space of the recipient eye, and capable of integrating the axons into said layers. Further aspects and embodiments are described below. [Brief description of the drawings]

[0055] The technology described herein may be more fully understood with reference to the following drawings, which are for illustrative purposes only.

[0056] [Figure 1A] 1 shows a diagram of a subretinal graft, according to certain aspects of the present disclosure. [Figure 1B] FIG. 1 shows a diagram of a bioprosthetic retinal patch comprising hPSC-derived retinal tissue (organoid) and a bioprosthetic scaffold support, according to certain embodiments. [Figure 1C] 1 shows a bioprosthetic retinal patch comprising multiple hPSC-derived retinal tissue fragments and a bioprosthetic scaffold support, according to certain embodiments. [Figure 1D] FIG. 1 shows a diagram of a bioprosthetic retinal patch comprising hPSC-derived retinal tissue (organoid), a bioprosthetic scaffold support, and an RPE component, according to certain embodiments. [Figure 1E] FIG. 1 shows a diagram of a bioprosthetic retinal patch comprising hPSC-derived retinal tissue, a bioprosthetic scaffold support, and a light-sensitive diode (photodiode) component, according to certain embodiments. [Diagram 2] A chart describing the Birmingham Eye Trauma Terminology System (BETTS) is shown. [Figure 3A] Images of hPSC-derived retinal tissue stained with an antibody specific for CALB2, a calretinin marker expressed in neurons including the retina. [Figure 3B] Images of hPSC-derived retinal tissue stained with an antibody specific for the retinal cytoplasmic marker recoverin (RCVRN) are shown. [Figure 3C] FIG. 1 shows grafting of FACS-sorted PR cells from retinal organoids (retinal tissue bioprosthetic grafts) compared to human fetal retina. [Figure 4] Figure 4A shows ICH images of retinal integration and maturation of hESC-derived retinal progenitor cells (hESC-RPCs) transplanted into the epiretinal space in a mouse model. As shown, the majority of human progenitor cells are early neuronal marker Tujl negative, and can be seen migrating and integrating into the host's retinal ganglion cell (RGC) layer or inner nuclear layer (INL). Figure 4B shows ICH images of implanted hESC-derived retinal progenitor cells migrating into a large area of ​​the host subretinal site. Figure 4C shows ICH images of implanted epiretinal hESC-RPCs cells integrating into the host's retinal ganglion cell (RGC) layer, inner plexiform layer, and inner nuclear layer (INL). [Diagram 5]FIG. 5A shows an image of the implantation of a retinal tissue bioprosthetic graft. FIG. 5B shows an ICH image of a stained hESC-RPC epiretinal graft in a rabbit eye. A portion of the human retinal organoid was stained with the human nuclear marker HNu to show the human retinal progenitor cells of the human retinal organoid implanted in the epiretinal space in a rabbit eye. This sample was also counterstained with DAPI. FIG. 5C shows an ICH image of a stained hESC-RPC epiretinal graft in a rabbit eye. A portion of the human retinal organoid was stained with the human nuclear marker HNu to show the human retinal progenitor cells of the human retinal organoid implanted in the epiretinal space in a rabbit eye. FIG. 5D shows an ICH image of a human retinal organoid in a large animal model (rabbit), demonstrating that the retinal organoids described herein can be delivered to the intraocular space of a rabbit (large eye animal model) using a glass cannula via a pars plana incision without damaging the eyeball. The eyeballs were successfully preserved and stained to show the location of human retinal cells. [Figure 6] 1 shows a schematic diagram and corresponding image of a shock tube, according to certain embodiments. [Figure 7] Figure 7A shows the risk curves for the retina. The curves show the probability of a given CIS injury at a particular blast strength (specific impulse in kPa-ms) (red=CIS 1; green=CIS 2; CIS 3; black=CIS 4). Figure 7B shows the risk curves for the optic nerve. The curves show the probability of a given CIS injury at a particular blast strength (specific impulse in kPa-ms) (red=CIS 1; green=CIS 2; CIS 3; black=CIS 4). [Figure 8] 13 is an OCT image of a hESC-derived retinal tissue graft after transplantation into the subretinal space of a large-eyed animal model (wild type cat). [Figure 9] Image of HNu antibody immunostaining of hESC-derived retina after transplantation into cat eye, showing the presence of the retinal graft in the correct location. [Figure 10]FIG. 10A shows an image of hESC-3D-derived retinal tissue (retinal organoid) excised from a dish prior to transplantation. FIG. 10B shows an image of excised hESC-3D-derived retinal organoid growing in a dish prior to transplantation. FIG. 10C shows an additional image of hESC-3D-derived retinal organoid growing in a dish. FIG. 10D shows an IHC image demonstrating that the hESC-3D-derived retinal tissue bioprosthetic graft in the eye of a blind immunodeficient rat is layered and overlapped after application. FIG. 10E shows an IHC image demonstrating that the hESC-3D-derived retinal tissue bioprosthetic graft is layered and overlapped. FIG. 10F shows an ICH image of the hESC-3D-derived retinal tissue bioprosthetic graft implanted in the eye of a blind immunodeficient rat with outer segment-like processes in the outer layer immediately adjacent to the rat RPE. [Figure 11] 1 shows ICH images demonstrating maintained retinal tissue viability after overnight transport in Hib-E at 4° C. Arrows highlight viable implanted human cells. [Figure 12] 12A-12C show images of the surgical team transplanting hESC-3D retinal tissue into the subretinal space of a wild type cat. FIG. 12D shows the intraocular pressure adjustment device and the RetCam device imaging the graft. FIG. 12E shows two ports inserted into the cat's eye for intraocular surgery. FIG. 12F shows the retinal detachment (bleb) for implanting the hESC-3D retinal tissue bioprosthetic graft into the subretinal space. FIG. 12G shows the cannula for injecting hESC-3D retinal tissue. FIG. 12h shows the hESC-3D retinal tissue in the subretinal space of a wild type cat imaged with the RetCam. FIG. 12I shows the location of the OCT image of the hESC-3D retinal tissue 5 weeks after transplantation placed in the subretinal space of a wild type cat. FIG. 12J shows a cross-sectional OCT image of the hESC-3D retinal tissue 5 weeks after transplantation placed in the subretinal space of a wild type cat. FIG. 12K shows a 3D reconstruction of the OCT images to estimate the overall dimensions of the graft. [Figure 13-1]Figure 13A shows a cat eye with a subretinal graft that was PFA fixed, cryoprotected, and OCT saturated in preparation for sectioning. Figure 13B shows a cross section of a cat eye frozen in OCT. Figure 13C shows a 16 μm thick section of a cat eye in OCT, showing the graft as a central retinal bulge. Figure 13D shows a magnified image of a region of the frozen section showing preservation of the hESC-3D retinal tissue graft. [Figure 13-2] FIG. 13E shows an IHC image of a cat retina section with a hESC-3D retinal tissue graft 5 weeks after transplantation into the subretinal space. The graft shows the presence of numerous CALB2 (calretinin) positive neurons, with the arrow pointing to a CALB2[+] axon connecting the human graft to the cat's ONL. FIG. 13E-G show images of the hESC-3D retinal tissue graft in the cat's subretinal space stained with HNu, Ku80, and SC121 human (but not cat) specific antibodies, respectively. These results demonstrate that indeed the human tissue has been transplanted into the correct location in the cat's subretinal space. FIG. 13H shows images stained with BRN3A (a marker for RGCs) and a human nuclear marker. The asterisk indicates the area with the marker in the main image and is magnified in the inset. These results indicate that some of the cells in the graft are maturing into RGCs. [Figure 13-3] Figures 13I to 13K show staining with antibodies specific for human (but not cat) synaptophysin (hSYP) and the axonal marker NFL (specific for both cat and human neurons) and show the presence of punctate staining (arrows), indicating possible synapses formed by human neurons integrating into cat neurons. [Figure 13-4] Figures 13L to 13M show staining with antibodies specific for human (but not cat) synaptophysin (hSYP) and the axonal marker NFL (specific for both cat and human neurons) and show the presence of punctate staining (arrows), indicating possible synapses formed by human neurons integrating into cat neurons. [Figure 14]Figures 14A and 14B show images of the human (but not cat) specific synaptophysin antibody hSYP (red), and calretinin (green), which stains both cat and human neurons. Figures 14C and 14D show lower magnification images and provide a schematic of a large piece of cat retina with a hESC-3D retinal tissue graft. [Figure 15] Figures 15A-C show images of calretinin[+] axons (arrows) connecting the feline INL to calretinin[+] human cells in the graft, and Figures 15D and 15E show images of mature-appearing calretinin[+] neurons in the graft and calretinin[+] axons found throughout the graft. [Figure 16-1] Figures 16A-C show images of staining of the hESC-3D retinal tissue graft edges in the cat subretinal space. SC121 human cytoplasmic specific antibody (red) and Ku80 human nuclear specific antibody (green) stain the human retinal graft but not the cat retina. These images show connectivity between the graft and the host. [Figure 16-2] Figures 16D and 16E show images showing axons from hESC-3D retinal tissue grafts wrapping around feline PR in the layer immediately adjacent to the graft (arrows), and some SC121+ human axons can be seen traversing the feline ONL (arrowheads). [Figure 17] Shown is a RetCam image of a retinal organoid implanted in a cat, taken immediately after transplantation into the subretinal space. [Figure 18] 18A and 18B show diagrams comparing the ocular anatomy of humans and cats. [Figure 19] 1 shows an example of a timeline for differentiation of retinal organoids according to certain embodiments. [Figure 20-1] Figures 20A-20E show images of retinal progenitor markers and early photoreceptor markers in hESC-derived retinal tissue. [Figure 20-2] Figures 20F-I show images of retinal progenitor markers and early photoreceptor markers in hESC-derived retinal tissue. [Figure 21] 1 shows images of hESC-derived retinal tissue bioprosthetic grafts being implanted into the subretinal space of a wild-type cat eye using a glass cannula following pars plana vitrectomy. [Figure 22] 1 shows an image of a subretinal bleb into which a hESC-derived retinal tissue bioprosthetic graft is implanted. [Diagram 23] Color fundus and OCT images taken 3 weeks after implantation of hESC-derived retinal tissue bioprosthetic grafts are shown. [Figure 24] Shown are images of Group 1 (+ prednisone, - cyclosporine A) cat retinal sections stained with antibodies specific for microglia and macrophages. [Diagram 25] Shown are images of cat retinal sections from group 2 (+ prednisone, + cyclosporine A), also stained with antibodies specific for microglia and macrophages. [Figure 26] Graph comparing the number of cells positive for microglia and macrophage cell markers in cat retina sections from Group 1 (+ prednisone, - cyclosporine A) and Group 2 (+ prednisone, + cyclosporine A). [Figure 27] Figure 27A shows an image of a Group 2 (+Prednisone, +Cyclosporin A) cat retinal section stained with an antibody specific for the photoreceptor marker CRX. Figure 27B shows an image of a Group 2 (+Prednisone, +Cyclosporin A) cat retinal section stained with a human specific antibody, HNu. Figure 27C shows an image of a Group 2 (+Prednisone, +Cyclosporin A) cat retinal section stained with antibodies against both CRX and HNu. [Figure 28]Figure 28A shows images of Group 2 (+ prednisone, + cyclosporine A) cat retinal sections stained with an antibody specific for the retinal ganglion cell (RGC) marker BRN3A. Figure 28B shows images of Group 2 cat retinal sections stained with both BRN3A and the human-specific marker KU80. Figure 28C shows images of Group 2 cat retinal sections stained with BRN3A, the human-specific marker KU80, and DAPI. [Figure 29] Figure 29A shows an image of a cat retina section stained with an antibody specific for the calretinin marker CALB2, which is expressed in neurons, including the retina. Figure 29B shows an image of IHC staining for the marker SC121. The antibody to SC121 is specific for the cytoplasm of human cells. Figure 29C shows an image of a cat retina section stained with antibodies specific for the markers CALB2, SC121, and DAPI. [Diagram 30] Figure 30A shows an ICH image of retinal graft axons (stained with an antibody specific for the CALB2 marker) extending towards the cat retina. Figure 30B shows an ICH image of a retinal graft stained with antibodies specific for the human cell markers HNu and CALB2, thereby depicting the graft from the cat retina. Figure 30C shows an ICH image of GABA positive staining of graft axons, indicating that the axons of the implanted tissue integrated into the recipient retina have differentiated to become neurons. [Figure 31A] Shown are OCT images of human ESC-derived retinal organoids within the sub- and epiretinal spaces of a CRX-mutant cat with retinal degeneration (RD). [Figure 31B] Shown are OCT images of human ESC-derived retinal organoids within the sub- and epiretinal spaces of a CRX-mutant cat with retinal degeneration (RD). [Figure 31C] Shown are OCT images of human ESC-derived retinal organoids within the sub- and epiretinal spaces of a CRX-mutant cat with retinal degeneration (RD). [Figure 31D]Shown are OCT images of human ESC-derived retinal organoids within the sub- and epiretinal spaces of a CRX-mutant cat with retinal degeneration (RD). [Figure 31E] Shown are OCT images of human ESC-derived retinal organoids within the sub- and epiretinal spaces of a CRX-mutant cat with retinal degeneration (RD). [Fig. 31F] Shown are OCT images of human ESC-derived retinal organoids within the sub- and epiretinal spaces of a CRX-mutant cat with retinal degeneration (RD). [Figure 31G] Shown are OCT images of human ESC-derived retinal organoids within the sub- and epiretinal spaces of a CRX-mutant cat with retinal degeneration (RD). [Diagram 32] 1 shows ICH images of bioprosthetic retinal grafts containing BDNF-expressing hESC-derived retinal tissue 5 weeks after application into the subretinal space of a wild-type cat eye. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0057] Detailed Description The bioprosthetic retinal graft (or device) described herein can be used to treat retinal degenerative diseases and disorders. For example, the bioprosthetic retinal graft can include stem cell-derived tissue or cells. In some embodiments, the bioprosthetic retinal graft can also include a carrier and scaffold suitable for implantation into the intraocular space of a subject's eye to form a bioprosthetic retinal patch. In certain embodiments, the bioprosthetic retinal patch can include multiple stem cell-derived tissue or cell fragments on the carrier or scaffold and can be used to treat large areas of retinal degeneration or damage.

[0058] The present disclosure relates to cell and / or tissue compositions, and methods of preparing the cell and / or tissue compositions, suitable for therapeutic use in slowing the progression of retinal degenerative disease, slowing the progression of retinal degenerative disease after traumatic injury, slowing the progression of age-related macular degeneration (AMD), preventing retinal degenerative disease, preventing retinal degenerative disease after traumatic injury, preventing AMD, restoring retinal pigment epithelium (RPE), photoreceptor cells (PRCs), and retinal ganglion cells (RGCs) lost due to disease, injury, or genetic abnormality, increasing RPE, PRCs, and RCGs, or treating RPE, PRC, and RCG deficiency in a subject.

[0059] The term "subject" as used herein includes, but is not limited to, humans, non-human primates, and non-human vertebrates including all mammals, such as wild, domestic, and livestock animals, e.g., cats, dogs, cows, sheep, pigs, horses, rabbits, rodents, e.g., mice and rats. In some embodiments, the term "subject" refers to males. In some embodiments, the term "subject" refers to females.

[0060] The terms "treatment," "treat," "treated," or "treating," as used herein, can refer to therapeutic treatment or prophylactic or preventative measures, the purpose of which is to prevent or slow (alleviate) an undesirable physiological condition, symptom, disorder, or disease, or to obtain a beneficial or desired clinical outcome. In some embodiments, the term may refer to both treatment and prevention. For purposes of this disclosure, beneficial or desired clinical outcomes include, but are not limited to, one or more of: amelioration of symptoms; attenuation of the severity of a condition, disorder, or disease; stabilization (i.e., not worsening) of the condition, disorder, or disease state; delay in onset or slowing of progression of a condition, disorder, or disease; alleviation of the condition, disorder, or disease state; and detectable or non-detectable remission (partial or complete), or improvement or amelioration of a condition, disorder, or disease. Treatment also includes eliciting a clinically meaningful response. Treatment also includes prolonging survival compared to the expected survival rate in the absence of treatment.

[0061] Retinal implant Aspects of the present disclosure provide compositions and methods for treating, restoring, and / or ameliorating vision loss caused by traumatic injury or disease in a subject by restoring retinal tissue to the damaged site. In certain embodiments, the present disclosure provides methods for delivering engineered retinal tissue to the affected site, for example, using a biocompatible absorbent matrix, scaffold, and / or carrier, to restore vision loss in a subject. In retinal tissue engineering and delivery applications, when there is a large area of ​​damaged tissue, it is beneficial to create a large amount of a biocompatible scaffold to which engineered retinal tissue is attached and place it in a controlled manner in the eye of the subject.

[0062] In one aspect, there is provided an implantable biological retinal patch or biological retinal prosthetic device derived from human pluripotent stem cells (hPSCs), human embryonic stem cells (hESCs) and / or tissue, and / or human fetal or adult retinal tissue, useful for restoring vision following extensive ocular and retinal occlusive injury in a subject, slowing the progression of retinal degenerative disease, slowing the progression of retinal degenerative disease following traumatic injury, slowing the progression of age-related macular degeneration (AMD), preventing retinal degenerative disease, preventing retinal degenerative disease following traumatic injury, preventing AMD, restoring retinal pigment epithelium (RPE), photoreceptor cells (PRCs), and retinal ganglion cells (RGCs) lost due to disease, injury, or genetic abnormality, increasing RPE, PRCs, and RCGs, or treating RPE, PRC, and RCG deficiencies.

[0063] Figure 1A shows a diagram of a subretinal graft implanted in the subretinal space of a subject's eye according to certain embodiments of the present disclosure. Figure 1B shows a diagram of a bioprosthetic retinal patch comprising hPSC-derived tissue (organoid) and a bioprosthetic scaffold support.

[0064] In one aspect, human pluripotent (or embryonic) stem cell-derived tissue (hPSC-derived retinal tissue or hPSC-3D retinal tissue) can be used for transplantation into the subretinal or epiretinal space of a subject's eye. hPSC-3D retinal tissue represents a major advance in vision restoration therapies, as retinal tissue produced from hESCs maintains its native ability to fully differentiate and reestablish synaptic connectivity with the recipient retina after transplantation. Small slices of hESC-3D retinal tissue may contain approximately 1,000-2,000, or 2,000-3,000, or 1,000-5,000, 3,000-10,000, or 5,000-100,000, or 50,000-500,000, or 100,000-1 million, or more, photoreceptors, the key light sensing cells. By placing many individual hESC-3D retinal tissue pieces in a single patch of ultra-thin biomaterial, it is possible to produce large and flexible (yet implantable) biological retinal tissue bioprosthetic patches for vision improvement. This retinal tissue vision correction product can reduce surgical errors because the grafts and patches described herein allow for precise and controlled placement of retinal tissue grafts.

[0065] In certain embodiments, an in vitro engineered three-dimensional retinal tissue resembling a flattened cylinder (i.e., a disk) comprises a central core of retinal pigment epithelial (RPE) cells, and radially outward from the RPE cell core, a retinal ganglion cell (RGC) layer, a second retinal neuron layer (corresponding to the inner nuclear layer of the mature retina), a photoreceptor (PR) cell layer, and an outer layer of RPE cells. Each of these layers may have cells that characterize the layer that are fully differentiated, and may optionally contain precursors of the differentiated cells that characterize the layer. For example, the RPE cell layer (or core) may contain RPE cells and / or RPE progenitor cells; the PR cell layer may contain PR cells and / or PR progenitor cells; the inner nuclear layer may contain second retinal neurons and / or precursors of second retinal neurons; and the RGC layer may contain RGCs and / or RGC progenitor cells. In some embodiments, the progenitor cells in the different layers described herein have the ability to fully differentiate after transplantation.

[0066] The terms "hPSC-derived 3D retinal tissue", "hPSC-derived 3D retinal organoid", "hPSC-3D retinal tissue", "in vitro retinal tissue", "hPSC-derived retinal tissue", "retinal organoid", "retinal spheroid", and "hPSC-3D retinal organoid" are used interchangeably in this disclosure and refer to three-dimensional aggregates derived from pluripotent stem cells that contain retinal tissue. hPSC-derived 3D retinal organoids develop most or all retinal layers (RPE, PR, inner retinal neurons (i.e., inner nuclear layer) and retinal ganglion cells), and show the onset of synapse formation and axon formation as early as approximately 4-8 weeks in certain organoids, becoming more prominent at approximately 3 or 4 months of hESC-3D retinal development. The 3D retinal organoids disclosed herein may express the LGR5 gene, an adult stem cell marker and a key member of the WNT pathway. Additionally, hPSC-derived 3D retinal organoids can be genetically engineered to transiently or stably express transgenes of interest to enhance differentiation and / or act as reporters and / or to enhance the neuroprotective properties of hPSC-3D-derived tissue constructs or cells derived from such tissue constructs.

[0067] Although the present disclosure refers to hESC-derived 3D retinal tissue, one of skill in the art will understand that any pluripotent cell (ES cell, iPS cell, pPS cell, ES cell from a parthenogenetic organism, etc.), as well as embryonic, fetal, and / or adult retina can be used as a source of 3D retinal tissue for the methods of the present disclosure.

[0068] As used herein, "embryonic stem cells" (ES) refer to pluripotent stem cells (embryonic, induced, or both) that are (1) derived from a blastocyst before the cells have substantially differentiated into the three germ layers (ES); or (2) alternatively obtained from an established cell line (iPS). Unless otherwise specified, the term includes primary tissues and established cell lines with phenotypic characteristics of ES cells, and the progeny of those cell lines with pluripotent phenotypes. The ES cells may be human ES cells (hES). Prototypic hES cells have been described by Thomson et al. (Science 282:1145 (1998); and U.S. Patent No. 6,200,806) and may also be obtained from any one of a number of established stem cell banks, such as the UK Stem Cell Bank (Hertfordshire, UK) and the National Stem Cell Bank (Madison, Wisconsin, USA).

[0069] As used herein, "pluripotent stem cells" (pPS) refer to cells that can be derived from any source and that, under appropriate conditions, can produce progeny of different cell types that are relatives of all three germ layers (endoderm, mesoderm, and ectoderm). pPS cells can have the ability to form teratomas in 8-12 week old SCID mice and / or form identifiable cells of all three lobes in tissue culture. Definitions of pluripotent stem cells include human embryonic stem (hES) cells (see, e.g., Thomson et al. (1998) Science 282:1145) and human embryonic germ (hEG) cells (see, e.g., Shamblott et al., (1998) Proc. Natl. Acad. Sci. USA 95:13726); embryonic stem cells of other primates, such as rhesus monkey stem cells (see, e.g., Thomson et al., (1995) Proc. Natl. Acad. Sci. USA 92:7844), marmoset stem cells (see, e.g., (1996) Thomson et al., Biol. Reprod. 55:254), stem cells generated by nuclear transfer techniques (U.S. Patent Application Publication No. 2002 / 0046410), and induced pluripotent stem cells (see, e.g., Yu et al., (2007) Science 318:5858);Takahashi et al., (2007) Cell 131(5):861). pPS cells may be established as cell lines and thus may provide a continuous source of pPS cells.

[0070] As used herein, "induced pluripotent stem cells" (iPS) refer to embryonic-like stem cells obtained by dedifferentiation of adult somatic cells. iPS cells are pluripotent (i.e., capable of differentiating into at least one cell type found in each of the three germ layers of the embryo). Such cells can be obtained from differentiated tissues (e.g., somatic tissues such as skin) and can be dedifferentiated by genetic manipulations that reprogram the cells to acquire embryonic stem cell characteristics. For example, induced pluripotent stem cells can be obtained by inducing the expression of Oct-4, Sox2, Kfl4, and c-Myc in somatic stem cells. Thus, iPS cells can be generated by retroviral transduction of transcription factors such as Oct-3 / 4, Sox2, c-Myc, and KLF4 into somatic cells such as fibroblasts, hepatocytes, and gastric epithelial cells. Yamanaka S, Cell Stem Cell. 2007, l(l):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); 111 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.

[0071] It will be understood that the methods of the present disclosure can use any of the following defined embryonic stem cells (such as hES cells), embryonic-like stem cells (such as iPS cells), and pPS cells. In particular, it will be understood that hESC-derived 3D retinal organoids / retinal tissues can be derived from any type of pluripotent cell.

[0072] In one exemplary method for inducing 3D retinal organoids, pluripotent cells (e.g., hESCs, iPS cells) are cultured in the presence of Noggin protein (e.g., at a final concentration of 50-500 ng / ml) for 3-30 days. Basic fibroblast growth factor (bFGF) is then added to the culture in addition to Noggin (e.g., at a final concentration of 5-50 ng / ml) and the culture is continued for an additional 0.5-15 days. The culture is then added with morphogens Dickkopf-related protein 1 (Dkk-1) and insulin-like growth factor-1 (IGF-1) (e.g., 5-50 ng / ml each) in addition to the existing Noggin and bFGF and the culture is continued for an additional 1-30 days. Dkk-1 and IGF-1 are then removed from the culture and fibroblast growth factor-9 (FGF-9) is added to the culture in addition to Noggin and bFGF (e.g., 5-10 ng / ml). Culture is continued in the presence of Noggin, bFGF, and FGF-9 until retinal tissue forms, for example, for 1-52 weeks. Additional methods for deriving 3D retinal organoids / tissues are described in WO 2017 / 176810, published October 12, 2017, and incorporated herein by reference in its entirety.

[0073] In some embodiments, organoids (hPSC-derived retinal tissue) may be separated before application. Organoids may be separated at about 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks after development or culture. In some embodiments, organoids may be separated after 10 weeks after development or culture. Organoids can be separated into their component cell types by suspension in solution or mechanically, for example, by glass rod, sieve, blade, hydrophobic or hydrophilic surface, or any other suitable means. In certain embodiments, cell composition is prepared from hESC-3D retinal tissue by dissociating hESC-3D retinal tissue with papain.

[0074] The organoids described herein, or developing or differentiating organoids, may be cultured and / or produced in non-adherent conditions or a combination of adherent and non-adherent conditions. In some embodiments, the organoids or developing organoids may be cultured and manipulated on a substrate, followed by culture in non-adherent conditions. In some embodiments, the organoids may be cultured and manipulated on a substrate, followed by culture in adherent conditions. In some embodiments, the organoids may be cultured and manipulated in non-adherent conditions, followed by culture in adherent conditions. In some embodiments, the organoids may be cultured and manipulated in non-adherent conditions, followed by culture in non-adherent conditions. In some embodiments, the organoids may be cultured and manipulated in non-adherent conditions, followed by culture in non-adherent conditions.

[0075] In certain embodiments, the bioprosthetic retinal graft comprises hPSC-derived organoids having dimensions of about 0.5 mm x 0.5 mm to about 2 mm x 2 mm. In other embodiments, the bioprosthetic retinal graft comprises hPSC-derived organoids having dimensions of about 0.5 mm to about 2 mm.

[0076] In certain embodiments, proprietary, cGMP grade hPSC lines that provide a replenishable source of stem cells that have been tested in clinical trials for human ocular cell therapy can be used.

[0077] In some embodiments, cell compositions suitable for therapeutic use may be prepared as cell therapy products containing cryopreserved stocks of cGMP-grade human retinal progenitor cells capable of delivering trophic support to degenerating retinal cells. Furthermore, as shown in Figures 3A-3C, the retinal tissue of organoids derived in dishes closely resembles human fetal retina, with nearly identical photoreceptor content (Figure 3C), making them an excellent and replenishable source of primary human retinal progenitor cells. Figure 3A shows an image of hPSC-derived retinal tissue stained with an antibody specific for the calretinin marker CALB2, which is expressed in neurons including the retina. Figure 3B shows an image of hPSC-derived retinal tissue stained with an antibody specific for the retinal cytoplasmic marker recoverin (RCVRN).

[0078] In one aspect, the implantable biological retinal prosthetic device comprises human pluripotent stem cell derived tissue (hPSC-3D retinal tissue or hPSC-derived retinal tissue or organoids), human embryonic stem cells (hESCs) and / or tissue, and / or human fetal or adult retinal tissue, and a biocompatible carrier or scaffold to form a bioprosthetic retinal patch.

[0079] In some aspects, the biomaterial carrier or scaffold or matrix or delivery vehicle may be a structure such as a sheet, emulsion, network, slurry, or solution. In some aspects, the biomaterial carrier may be electrospun, printed, bonded, coated, lyophilized, or crosslinked. The biomaterial carrier or scaffold or matrix may include multiple structures or features such as fibers, ridges, microneedles, and / or other structural features. The biomaterial carrier may be composed of biocompatible materials such as polyphosphazenes, polyanhydrides, polyacetals, polyorthoesters, polyphosphoesters, polycaprolactones, polyurethanes, polypeptides, polycarbonates, polyamides, polysaccharides, polyamino acids, other polymers, proteins, metals, or ceramics. In some aspects, the biomaterial carrier may be composed in whole or in part of a derivative of a hyaluronan-based hydrogel, such as HYSTEM® hydrogel (BioTime, Inc.). In some embodiments, the biomaterial carrier or scaffold may include a combination of the aforementioned features and materials. In some embodiments, the carrier or scaffold may comprise a thermoreversible material and / or a shape memory metal. The scaffold (and bioprosthetic retinal patch) may be of any shape suitable for delivery of hPSC tissue and / or cells and / or other components, such as exosomes or trophic factors.

[0080] The biological scaffold or support may include, for example, an electrospun polymer. In one embodiment, the electrospun polymer scaffold has characteristics of Brunch's membrane. In some aspects, the thin electrospun nanofibers of the biomaterial include derivatives of HYSTEM® hydrogel (BioTime, Inc.).

[0081] In some embodiments, a biomaterial carrier or scaffold may be used that possesses all of the necessary characteristics for successful delivery and / or in situ immobilization of complex and fragile cells and macromolecules.

[0082] Recently, a family of hyaluronan-based hydrogels (brand names HYSTEM® and RENEVIA®) that mimic the natural extracellular matrix environment (ECM) has been developed for 3D cell culture, stem cell growth and differentiation, tissue engineering, regenerative medicine, and cell-based therapy. HYSTEM hydrogels were designed to reproduce the minimal composition required to obtain a functional extracellular matrix. Each component of the hydrogel can be crosslinked in situ and seeded with cells prior to in vivo injection, without compromising either the cells or the recipient tissue.

[0083] The technology underlying HYSTEM® hydrogels is based on a unique thiol cross-linking strategy to prepare hyaluronan-based hydrogels from thiol-modified hyaluronan and other ECM components. This has led to the development of a unique family of biocompatible, absorbable hydrogels. The basic elements of HYSTEM® hydrogels are hyaluronan and gelatin, each of which is thiol-modified by carbodiimide-mediated hydrazide chemistry. Hydrogels are formed by cross-linkable mixtures of these thiolated macromolecules with polyethylene glycol diacrylate (PEGDA) (see U.S. Pat. Nos. 7,928,069 and 7,981,871, which are incorporated herein by reference in their entirety). The gelation rate and the firmness of the hydrogels can be controlled by varying the amount of cross-linker. One attribute of these hydrogels is their high water content of over 98%, resulting in high permeability to oxygen, nutrients, and other water-soluble metabolites.

[0084] Hydrogels such as HYSTEM® have been shown to support the attachment and growth of a wide variety of cell types and tissues in both 2D and 3D cultures, and have shown a high degree of biocompatibility upon in vivo implantation in animal studies. These hydrogels are readily degraded in vitro and absorbed by collagenase and hyaluronidase enzyme-mediated hydrolysis in vivo. When implanted in these hydrogels, cells remain attached and localized within the hydrogel, but gradually degrade the implanted matrix and replace it with the cells' natural ECM.

[0085] Crosslinkers can include, for example, bifunctional, trifunctional, and multifunctional molecules that react with thiols (e.g., maleimide groups), oxidizing agents that initiate crosslinking (e.g., GSSG), glutaraldehyde, and environmental influences (e.g., heat, gamma / e-beam). In some embodiments, no crosslinker is required.

[0086] Specific examples of hydrogels suitable for providing absorbent matrices for use in the embodiments of the present disclosure are described, however, it will be understood that any suitable biocompatible matrix may be used, for example, gels made using oxidized glutathione (GSSG) as a crosslinker may be used (see U.S. Patent Application Publication No. 20140341842, incorporated herein by reference in its entirety).

[0087] The carrier or scaffold may be composed of decellularized tissue, such as retinal tissue. The decellularized tissue may be intact, disrupted, engineered, or mature tissue. The bioprosthetic retinal implant may be composed entirely or partially of pieces of human embryoid body retina, or fetal retinal tissue, or adult retinal tissue, or organoid cells or other, or biomaterials, or a combination thereof.

[0088] The cell, tissue, and biocompatible carrier, matrix, and scaffold compositions described herein induce growth of the applied tissue, such that the therapeutic results may be long-lasting, for example, beyond 18 months. In some embodiments, the carrier or scaffold is permeable to nutrients, trophic factors, and oxygen.

[0089] In some embodiments, the bioprosthetic carrier or scaffold may serve as both a cell culture and a delivery matrix.

[0090] In some embodiments, the bioprosthetic retinal patch has dimensions of about 0.5 mm x 1 mm x 1 μm to 8 mm x 12 mm x 100 μm in length x width x thickness. In some embodiments, the bioprosthetic retinal patch has dimensions of about 2 mm x 4 mm x 50 μm in length x width x thickness. In other embodiments, the bioprosthetic retinal patch has dimensions of about 4 mm x 6 mm x 10 μm in length x width x thickness. In some embodiments, the area of ​​the bioprosthetic retinal patch is about 3 mm x 6 mm, about 4 mm x 6 mm, about 4 mm x 5 mm.

[0091] In some aspects, the bioprosthetic retinal graft or patch may be anchored using any suitable material after implantation.

[0092] In one aspect, the retinal tissue and the biocompatible scaffold are attached to one another with a biocompatible adhesive.

[0093] In another aspect, the cell therapy is prepared by a method comprising embedding organoid pieces in biocompatible scaffold, wherein biocompatible scaffold is initially prepared in liquid form, then forms gel, but before completely solidifies, the pieces are placed in the liquid scaffold, so that when scaffold gels, organoid pieces are embedded in gel.In one embodiment, graft can be applied before scaffold gels completely.In another embodiment, graft can be applied in suspension of biomaterial, or together with biomaterial or biocompatible adhesive, or combination thereof.

[0094] In some embodiments, the organoids may be crosslinked to a biocompatible scaffold by natural proteins or small molecule crosslinkers such as integrins or fibronectin. In some aspects, pieces of retinal tissue are fixed or attached to a large biomaterial scaffold to create a large retinal implant or biological retinal prosthetic device.

[0095] In some embodiments, the organoids may be modified to increase their adhesion to a carrier, substrate, or recipient tissue.

[0096] In some aspects, pieces of retinal tissue are fixed or attached to a thin film of biomaterial to create an implant or biological retinal prosthetic device, as shown in Figure 1 C. In some aspects, the thin film of biomaterial may contain biological components such as RPE layers, RPE sheets, RPE cells, progenitor cells or cell types other than those that comprise the organoids, as shown in Figure 1 D.

[0097] In some aspects, the organoids or biological components can be cultured or attached to a non-biodegradable carrier or scaffold that is enzymatically dissolved, and the retinal tissue and / or other biological components can be attached to the biodegradable carrier or scaffold and implanted.

[0098] In certain aspects, the retinal tissue and biological scaffold may be described as an implant. In certain aspects, the retinal tissue and biocompatible carrier or scaffold may be described as a medical device or a biological retinal prosthetic device.

[0099] In some aspects, multiple three-dimensional (3D) retinal tissue slices, each carrying about 1,000-2,000, or 2,000-3,000, or 1,000-5,000, or 3,000-10,000, or 5,000-100,000, or 50,000-500,000, or 100,000-1 million photoreceptors, can be placed in a thin or ultra-thin flexible biomaterial, which captures and transmits visual information synaptically (or by other means) to the subject's RGCs, which then transmit the information to the subject's visual cortex. The implanted whole tissue fragment can produce a patch or biological retinal prosthetic device that can create a wide visual angle (up to 30° depending on the dimensions of the biological retinal patch) with approximately 1,000-2,000 or 2,000-3,000, or 1,000-5,000, 3,000-10,000, or 5,000-100,000, or 50,000-500,000, or 100,000-1 million or more individual light sensors, i.e. photoreceptors, supporting useful and functional vision. In comparison, the ArgusII neuroprosthetic device has only 60 sensors, and even when precisely placed in the subretinal space, the recipient can only distinguish the shape of objects.

[0100] In some embodiments, the organoid can be combined with synthetic materials, sensors, chips, or electronic devices. In one embodiment, a bioprosthetic retinal patch is described, comprising hPSC-derived retinal tissue and a film or biological scaffold or matrix that comprises a biocompatible material with a light-sensitive diode (photodiode) that forms a light-sensitive component or layer. The hPSC-derived retinal tissue or organoid is combined with or attached to the light-sensitive layer using any of the materials and methods described herein. Figure 1E shows a diagram of a bioprosthetic scaffold with a photodiode. The photodiode layer can enhance the response to light (light capture, light conversion to electrical signals and signal transmission) by the host's remaining functional photoreceptors and the retinal tissue components of the patch, especially the area where the retinal graft tissue is still developing or differentiating.

[0101] In other embodiments, large grafts containing multiple hESC-3D retinal tissue fragments and a biocompatible scaffold are implanted into the subretinal space of a subject, resulting in synaptic integration without tumors. In some embodiments, the biocompatible scaffold is porous, which allows for easier synaptic connections and molecular movement between cells and cell layers.

[0102] Therapeutic targets of such technology are human RD conditions associated with PR death and blindness, including but not limited to retinitis pigmentosa (RP) and age-related macular degeneration (AMD). Cone-only hPSC-3D retinal tissue can be derived from retinal organoids to treat disorders or diseases such as AMD. Bioengineered chips (e.g., SecondSight, ARGUS® Π, 60 pixels) work similarly, but the performance of biological designs may exceed that of electronic designs due to the limitations of electronics and the short lifespan of implanted electronic chips. Biological retinal patches can be integrated into the host's tissue, carry thousands of PRs (i.e., pixels) per retinal organoid slice, and be tailored (constructed) to treat individual diseases.

[0103] In certain aspects, intraocular implantation can be performed by any acceptable method, for example, by the methods described in WO 2016 / 108219, which is incorporated by reference in its entirety.

[0104] In other embodiments, intraocular implantation can be performed using a mechanically powered delivery device, such as the UMP3 UltraMicroPump III with Micro4 Controller (World Precision Instruments) or modifications thereof, following the manufacturer's instructions.

[0105] In certain embodiments, the delivery device may include a cannula. The cannula may have an inner diameter of about 0.5 mm to about 2.5 mm, or about 1 mm to about 2 mm, or about 1.12 mm. The cannula may also have an outer diameter of about 0.5 mm to about 3 mm, or about 1 mm to about 2.5 mm, or about 1.25 mm to about 1.5 mm, or about 1.52 mm.

[0106] In certain embodiments, the bioprosthetic retinal graft or patch may be delivered to the subject's intraocular space using a cannula, in which air bubbles are introduced before and / or after the bioprosthetic retinal graft or patch to prevent it from exiting the cannula before it is in place, as shown in FIG. 1G. In certain embodiments, intraocular pressure may be applied to the subject's eye simultaneously with implantation of the bioprosthetic retinal graft or patch to help the bioprosthetic retinal graft or patch stay in place after implantation. In another embodiment, epinephrine may be injected into the vitreous space to reduce bleeding that may result from application of the bioprosthetic retinal graft or patch using an incisional procedure, such as a retinotomy.

[0107] In certain embodiments, surgical procedures may include, but are not limited to, vitrectomy, tension-reducing vitrectomy, tension-reducing retinotomy, use of retinal tacks, retinal detachment and macular translocation. In the clinic, tension-reducing retinotomy is used, which allows large pieces of the patient's retina to be detached and reattached. The purpose of these procedures may be to place a large bioprosthetic retina in the subject's subretinal space, allowing a large area of ​​the subject's eye to regain visual perception. In certain embodiments, adhesives, staples, or any other suitable material may be used to apply or fix the bioprosthetic retinal grafts and patches described herein and / or aid in the healing of the surgical wound.

[0108] In certain aspects, the bioprosthetic graft or patch can be rolled or otherwise compressed to fit into a smaller incision (about 3 mm or less). The graft or patch can then unfold or expand in situ, as shown in FIG. IF, to return to its original shape. In some embodiments, the graft or patch can return to its original shape upon implantation into the subject's eye without further surgical intervention or manipulation. In some embodiments, the graft or patch can return to its original shape on its own within about 2-15 seconds after implantation without further manipulation. In certain embodiments, the graft or patch can be preloaded and / or stored in a delivery device for a period of time prior to delivery into the subject's eye.

[0109] In certain aspects, several bioprosthetic retinal grafts or patches may be loaded into a delivery device that includes a delivery component, such as a cannula, which are applied one after the other to the intraocular space to cover a large area.

[0110] The aspects of the present disclosure provide a robust visual restoration therapy for patients, especially those whose retina is too damaged to be preserved by neuroprotection alone, in which individual photoreceptors can permanently synapse with recipient ganglion cells and / or other retinal or supporting cells, and can provide wide-angle visual recovery or visual relief within 12 months after transplantation. This visual restoration method is highly efficient and permanent, since individual sensors (photoreceptors) synapse with the target's RGCs. In contrast, subretinal implanted artificial neuroprosthetic devices gradually reduce contact with RGCs in retinal damage areas where the retina remains largely intact but is subject to gradual irreversible degeneration, for example after blast injury or degenerative disease.

[0111] As used herein, the term "synaptic activity" or "synaptically" refers to any activity or phenomenon that is characteristic of the formation of a synapse between two neurons.

[0112] The therapeutic efficacy of the bioprosthetic grafts and methods of making bioprosthetic grafts described herein can be measured, for example, by an increase in visual evoked potentials (VPEs) (e.g., at selected time points after blast injury), which is a reliable method of assessing the strength of visual signals reaching the brain. Electroretinography, multifocal ERG, multielectrode array (MEA) and / or RetiMap techniques can also be used.

[0113] In some embodiments, the use of advanced methods such as genetic transsynaptic tracers, WGA-HRP (expressed by the implant but not the recipient retina), WGA-Cre, human SYP, SC121 antibody or immunoelectron microscopy to assess synaptic connectivity between the graft (hPSC-3D retinal tissue and / or cells, etc.) and / or bioprosthetic retinal patch (hPSC-3D retinal tissue and / or cells, etc., and a biocompatible carrier or scaffold) and the recipient retina is provided to demonstrate chimeric (graft:recipient) synaptic connectivity. This tracing not only helps to track graft-host attachment, but also allows for the distinction between cell fusion and neuroprotection and specific synaptic integration.

[0114] In some embodiments, large-eyed animal models, such as Pde6a- / - dogs, Aipl- / - cats, Cngb3-mutant dogs, and Crx-mutant [+ / -] cats, Aipl-1 mutant cats, or blast-induced intraocular injury rabbits, each with PR degeneration, retinal degeneration and / or optic nerve degeneration similar to human subjects with genetic retinal degenerative conditions, retinal diseases, or retinal damage, can be used to assess the efficacy of hPSC-3D retinal tissue or hPSC-3D bioprosthetic retinal implants / grafts.

[0115] In some embodiments, in addition to behavioral testing, in vivo readout approaches such as, but not limited to, full-field ERG, multifocal ERG microelectrode arrays (MEAs), pupil imaging, and visual evoked potentials (VEPs) can be used to assess the degree of visual recovery following transplantation of hPSC-3D retinal tissue into the subretinal space of a subject.

[0116] In some embodiments, subretinal grafts of hPSC-3D retinal tissue (retinal organoids; bioprosthetic retinal implants / patches) can act as biological analogs of neuroprosthetic devices, capturing and transmitting visual information synaptically to retinal ganglion cells and then to the visual cortex. In another embodiment, the implant supports the restoration of visual perception (light detection) in a subject.

[0117] In yet another embodiment, the hPSC-derived retinal organoid bioprosthetic implant / patch or biological retinal prosthetic device carrying PR layer and secondary neuron layer provides light sensors that can synaptically transmit visual information to the subject's RGCs, which remain even after all PRs have degenerated. Unlike electronic prosthetic chips, the hPSC-derived retinal organoid-based "bioprosthetic" implant can allow long-lasting synaptic integration and can also be adjusted to carry more cones than rods to repair and reconstruct the macula. In some embodiments, long-term photosensitivity recovery can be seen in the majority of subjects using subretinal transplanted hPSC-3D retinal tissue.

[0118] In some embodiments, synaptic connectivity and functional integration of the subject's retinal circuitry with the hPSC-3D retinal tissue grafts can be demonstrated by pre-embedding immuno-EM, electroretinogram recordings, and multi-electrode array recordings.

[0119] In some embodiments, tumor-free engraftment of hESC-3D retinal tissue transplanted into the subretinal space occurs over at least about 6-24 months, with lamination and development of the PR and RPE layers, including PR outer segment elongation, synapse formation, electrophysiological activity, and connectivity with recipient retinal cells, and development into a more mature retinal immunophenotype. In some embodiments, hESC-3D retinal tissue grafts improve the subject's visual perception within about 5-10 months after transplantation, in part due to slow maturation and synaptic integration. In some embodiments, cytoplasmic fusion of the graft with the host is demonstrated in addition to specific synaptic connectivity between the graft and the host.

[0120] Transplantation of fetal retina into the subretinal space of visually impaired patients has resulted in improved vision in up to 7 out of 10 cases. Although it may be reasonable to assume that fetal retinal grafts positively influence the degenerating patient retina through neuroprotective mechanisms, there is also evidence that specific synaptic connectivity is established between the graft and the recipient retina. In both RD rats and RD patients, human fetal retinal grafts have been found to improve visual responses (superior colliculus activation in rats, improved vision in patients [ClinicalTrials.gov ##NCT00345917, NCT00346060]).

[0121] Similarly, the hPSC-3D retinal tissue of the present disclosure was found to enable light-induced superior colliculus responses in blind RD rats that lacked functional PRs, indicating that the grafted PRs transmitted visual information to the brain. There is also evidence that hPSC-3D retinal organoids develop PR inner / outer segments and cilia, even though the grafts did not maintain a continuous laminated structure during subretinal grafting. The hPSC-3D retinal tissue closely resembles the human fetal retina, exhibiting robust synapse formation and electrical activity after approximately 6-8 weeks of development, and containing rudimentary inner segment-like processes immunopositive for peanut agglutinin (PNA), collectively indicating that the tissue rapidly develops, matures, and synaptically integrates when grafted subretina. Thus, evidence is provided herein of graft / host binding of hPSC-3D retinal tissue grafted into the subretinal space of immunosuppressed wild-type cats. Taken together, these data indicate that hPSC-derived 3D tissues and bioprosthetic grafts can restore retinal photosensitivity, at least at the grafted site.

[0122] One advantage of this approach is the ability to induce human fetal-like retinal tissue that itself carries an RPE layer. This RPE layer can support the engraftment of hPSC-3D retinal tissue after transplantation. Competing techniques can generate neural retinal layers but not RPE from hPSC cultures. The neural retina and RPE arise together, induce each other's structural and functional maturation during development, and depend on each other to perform visual function. Transplantation of hPSC-derived neural retina without an RPE layer deprives the developing PR of the paracrine and structural support of the RPE. Gaps may develop between the RPE layer of the recipient retina and the PR of the graft in the subretinal space. Lack of physical interaction between the RPE microvilli and the developing PR may inhibit the ability of the apical RPE to induce PR outer segment outgrowth. Alternatively, the hPSC-3D retinal tissue obtained by the method described herein is not dependent on the proximity to the recipient's RPE, and is highly engrafted and differentiated (as an independent patch) in the subretinal graft. This increases the ability of the hESC-3D retinal tissue patch to restore visual function. There is also evidence that both retina and transplanted RPE provide better visual improvement in RD patients. However, these pilot trials use human fetal retinal tissue, which cannot be used in routine treatment due to ethical restrictions and tissue availability. Human ES cells provide an unlimited cell source for deriving retinal tissue. Thus, the hPSC-3D retinal tissue graft of the present disclosure overcomes the two major obstacles to the treatment of retinal degenerative diseases and injuries: the availability of human fetal retina and ethical restrictions.

[0123] Novel "sensors" are needed that can electrically couple to the subject's remaining retina and enable the transmission of electrical signals to restore light perception to PR-degenerated retinas. Human ESC-derived retinal tissue (retinal organoids, 0.3-0.5 mm in size) resembles human fetal retina (histologically and based on marker expression) and, when grown as substrate-attached aggregates, develops layers of RPE, PR, second-order retinal neurons, and RGCs by 6-8 weeks of in vitro development. When grown as substrate-attached aggregates, hPSC-3D retinal tissue develops axons (especially RGC-specific long axons) and multiple synaptic boutons by 6-8 weeks of development. This hPSC-3D retinal tissue can also become increasingly electrically active between weeks 8-12 of in vitro development. A single piece of retinal organoid transplanted into the subretinal space can result in a sufficient number of PRs such that blind animals can restore light perception.

[0124] Neurotrophic factors are a diverse group of soluble proteins (neurotrophins) and neurogenic cytokines that support the growth, survival, and function of neurons. Neurotrophic factors can activate multiple pathways in neurons, mitigate neurodegeneration, preserve synaptic connectivity, and inhibit cell death in retinal tissue. If neuroprotection is provided in the form of small molecules, neuroprotective proteins such as brain-derived neurotrophic factor (BDNF), or cells, and delivered efficiently and rapidly enough to arrest cell death and / or the onset of retinal remodeling and scarring, the acutely damaged retina will survive. However, if degeneration continues untreated, progressive vision loss may be predicted due to loss of photoreceptors, RGCs, and other retinal neurons, as well as retinal remodeling and scarring.

[0125] The retina is a thin layer of extremely sensitive neural tissue that receives light stimuli and converts them into electrical impulses that are transmitted via the optic nerve to the brain (lateral geniculate nucleus) and ultimately to the visual cortex. The optic nerve originates in the retina, which is formed by the axons of retinal ganglion cells (RGCs), one of seven cell types found in retinal tissue. Contusion injuries occur when the eyeball is first compressed by the force of the blast and then extends beyond its normal shape to more distal locations as it returns to its normal shape. Thus, non-penetrating eye injuries are common in combat zones and can result in retinal trauma, such as retinal detachment, optic nerve breakage, retinal remodeling, and axonal deafferentation (disruption of the afferent connections of nerve cells), which often leads to slow cell death (up to several months) and progressive vision loss, even if the retinal structure is initially preserved.

[0126] In some embodiments, the hESC-derived retinal tissue graft can deliver neurotrophic factors and / or mitogens after implantation. In some embodiments, the hESC-derived retinal graft or patch containing cells dissociated from the hESC-derived retinal tissue can also deliver neurotrophic factors and / or mitogens after implantation. In some embodiments, the hESC-derived retinal tissue and / or cells can deliver neurotrophic exosomes to the subject after implantation. The neurotrophic factors and mitogens that the grafts described herein can deliver to the subject include, but are not limited to, brain-derived neurotrophic factor (BDNF), glial-derived neurotrophic factor (GDNF), neurotrophin-34 (NT34), neurotrophin 4 / 5, nerve growth factor-beta (βNGF), proNGF, PEDF, CNTF, the pro-survival mitogen basic fibroblast growth factor (bFGF=FGF-2), and pro-survival members of the WNT family.

[0127] The current military standard of care for ocular injuries due to trauma or blast overpressure injury employs the Birmingham Eye Trauma Terminology System (BETTS) and Ocular Trauma Classification Group to determine appropriate treatment (see Figure 2). Blast injuries are broadly attributable to four mechanisms: primary blast (overpressure impulse); secondary effects, such as penetrating injuries from fragments blown off by the force of the blast; tertiary injuries, such as when a person strikes a solid structure; and quaternary injuries, which are caused by concomitant processes such as toxic fumes, chemical burns, or long-term psychological effects (Morley et al. 2010). Closed ocular trauma is divided into several zones, each with a unique injury pattern. Zone I includes the conjunctiva and corneal surface, while Zone II includes the anterior chamber, lens, and ciliary folds. Zone III includes the retina and optic nerve. The zones are illustrated in Figure 3.

[0128] There are several tested guidelines for the retina / optic nerve response to high-pressure blast injury. If the primary disruption in zone III is retinal detachment, rapid apoptosis of the photoreceptor layer begins days to weeks after injury, followed by degeneration of the inner nuclear layer (INL), retinal remodeling, visual distortion, and visual loss. However, the retinal ganglion cell (RGC) layer survives for months to years after injury as long as axonal connectivity between RGC nerve fibers (forming the optic nerve) and neurons in the visual cortex is preserved.

[0129] The viability of RGCs depends on their connectivity with visual cortex neurons, and the aforementioned afferent fibers transport supportive (trophic) factors between RGCs and visual cortex neurons. Blast exposure can cause deafferentation, thus cutting off the flow of trophic factors, leading to a slow but steady loss of vision. Restoration of trophic support (even partially) leads to the preservation of RGCs. Several trophic factors administered together can provide a potent neuroprotective defense against RGC apoptosis after axotomy. Therefore, it is useful to perform treatments to preserve RGCs after connectivity loss, days to weeks after injury.

[0130] Photoreceptor viability may depend, in part, on trophic support from, for example, the retinal pigment epithelium (RPE) and synaptic contacts with inner nuclear layer (INL) neurons. Photoreceptor viability and function depend on RPE-photoreceptor connectivity. Retinal detachment after blast injury results in degeneration of photoreceptor outer segments. The period during which photoreceptor function can be restored after reattachment is usually several days to several weeks after injury. As shown herein, restoration of trophic support (even partial) to photoreceptor cells leads to long-term preservation of photoreceptors.

[0131] Efficient treatment of visual problems associated with blast ocular injuries requires a neuropathological understanding of the damage caused to the visual system by blast injury. While the initial damage may not be immediately apparent, the blast pressure wave causes stretching and / or tearing of cells and shearing of axons in the direction of wave propagation, which slowly degenerates the retina and optic nerve. The multi-trauma nature of combat injuries often results in competing priorities for treatment. In the field, the primary concern is blood loss and resuscitation, but once the condition has stabilized, attention can be directed to ensuring the best overall outcome for the injury. Ocular treatment is often initiated hours to days after injury. This therapeutic window is well within the time frame believed to allow for effective treatment options for closed ocular injuries. Preserving the original neural architecture of the retina required for visual function and preventing retinal degeneration (by neuroprotection) after blast injury are viable therapeutic mechanisms to mitigate blindness.

[0132] Thus, in one embodiment, a cell composition (hESC-3D retinal organoid) suitable for therapeutic use prepared from hPSC-3D retinal tissue is obtained and transplanted into the intraocular space of a subject, where the cells can secrete extracellular components such as neurotrophic factors, mitogens, and / or exosomes. In some embodiments, the cell composition delivers trophic factors (by secretion or other mechanisms) continuously during the appropriate therapeutic window. In some embodiments, the cell composition delivers (by secretion or other mechanisms) a combination of several trophic factors, mitogens, and / or exosomes, and other extracellular components at the same time. In another embodiment, the trophic factors, mitogens, and / or exosomes, and other extracellular components produced by a bioprosthetic retinal graft or patch transplanted into the intraocular space (e.g., subretinal or epiretinal) can provide a potent neuroprotective defense against retinal cell death. Therapeutic targets can include some or all cell types of the subject's retina (e.g., photoreceptors, RPE, secondary neurons, RGC / optic nerve).

[0133] In some embodiments, the therapeutic effect is enhanced by transplanting cell compositions that include RPE cells, retinal ganglion cells (RGCs), second-order retinal neurons (corresponding to the inner nuclear layer of mature retina), and photoreceptor (PR) cells. The therapeutic effect can be enhanced by the combination of neuroprotection from transplanted cells. In other embodiments, different cell types can be separated and isolated, thereby enriching certain cell types and therefore enriching certain trophic factors to treat certain diseases, injuries, or conditions.

[0134] The stem cell-derived grafts described herein can provide long-lasting trophic support to degenerating retinal neurons and may therefore be a broadly applicable therapeutic modality for blast injury. Retinal cell grafts can reduce vision loss following blast injury in zone III (retina-optic nerve-visual cortex).

[0135] In one embodiment, grafts of stem cell-derived human retinal progenitor cell compositions were prepared to exert strong neuroprotective support on the neural retina and optic nerve of rabbits damaged by blast injury of CIS 2-3, thereby alleviating visual loss. The functional integrity of some transplanted neurons can further protect the retina from degeneration, which may be a positive factor for visual preservation.

[0136] In other aspects, the cell compositions or stem cell-derived grafts can provide long-lasting trophic support to degenerating retinal neurons, providing a feasible and widely applicable therapeutic intervention to attenuate vision loss due to blast injury in the eye. The cell therapy compositions described herein can positively impact the preservation of photoreceptors and retinal ganglion cells (RGCs).

[0137] In certain aspects, the therapeutic cell compositions described herein provide efficient, controlled, continuous paracrine delivery of a mixture of neurotrophic factors to damaged retinal tissue. The therapeutic cell compositions described herein may be particularly effective in retinal injury where the retina remains largely intact following blast injury, but is prone to gradual, irreversible degeneration due to disruption of higher order tissue architecture.

[0138] Figures 5B-5D demonstrate that subretinal grafts of human retinal progenitor cells differentiated from human embryonic stem cells (hESCs) can be successfully transplanted into the intraocular space of a large-eyed animal model (rabbit), preserving the retinal layer thickness of the adult mammalian retina for up to 3 months, without detrimental effects on the recipient retina, and without tumor formation. Cells from these grafts migrate and integrate into the recipient retinal layer to strengthen the recipient retina. Such cells can intermingle with recipient retinal cells in the RGC and INL, and provide paracrine support to surrounding host cells. Figure 4A shows ICH images of retinal integration and maturation of hESC-derived retinal progenitor cells (hESC-RPCs) transplanted into the epiretinal space of a mouse model. As shown, the majority of human progenitor cells are negative for the early neuronal marker Tujl, and can be seen to have migrated and integrated into the host retinal ganglion cell (RGC) layer or inner nuclear layer (INL). Figure 4B shows an ICH image of implanted hESC-derived retinal progenitor cells migrating into a large area of ​​the host subretinal site. Figure 4C shows an ICH image of implanted epiretinal hESC-RPC cells integrating into the host retinal ganglion cell (RGC) layer, inner plexiform layer, and inner nuclear layer (INL). Cells attached within the subretinal and epiretinal spaces are able to migrate into the host retina without leaving a bulge in the subretinal space or epiretinal membrane above the RGC layer.

[0139] In one embodiment of the present disclosure, neuroprotection by cells transplanted into a blast-injured retina increases cell viability and / or cell survival rate by about 10% to about 250% compared to cell viability in a control retina.

[0140] The cell compositions described herein are suitable for therapeutic applications to sustain viability and visual function of the retina, optic nerve, and visual cortex following retinal detachment and optic nerve damage due to closed wounds or disease of the eye. Because this technology does not require an autologous donor cell source, therapeutic cells can be made available on demand for the treatment of ocular trauma, disease, and vision loss.

[0141] In some embodiments, by 3-6 months after implantation, 80% of subjects have viable retinal cells in the subretinal / epretinal spaces. In another embodiment, 80% of subjects with retinal grafts found by OCT (about 64% of all subjects) will have improved VEP and ERG outcomes by 1, 2, 3, 4, 5, or 6 months after intraocular implantation of the bioprosthetic retinal graft or patch, due at least in part to neuroprotection from retinal progenitor cells.

[0142] In one embodiment, the preservation of retinal thickness in the subject occurs from about 1 to about 6 months after transplantation. In another embodiment, the subject has reduced cell death at or near the graft, as determined, for example, by Cleaved Caspase-3, γH2AX (early apoptosis markers), and Tunnel staining (late marker).

[0143] In yet another embodiment, preservation of retinal thickness (as a primary readout of retinal degeneration) occurs in at least about 64% of subjects from about 1 to about 6 months post-transplant, and cell death is reduced as determined by Cleaved Caspase-3, γH2AX (an early apoptosis marker), and Tunnel staining (a late marker).

[0144] Subretinal grafts can provide neuroprotection to the photoreceptors and outer plexiform (synaptic) layer, while epiretinal grafts can provide neuroprotection to the RGCs / optic nerve, second order retinal neurons, and inner plexiform (synaptic) layer.

[0145] In one embodiment, subjects exhibited about 1% to about 15% retinal thickness preservation about 6 months after implantation of the bioprosthetic graft.

[0146] In certain embodiments, the therapeutic cell composition is applied with or without an immunosuppressant.

[0147] The retina is a complex structure and preservation of cellular and synaptic networks is beneficial for maintaining vision. Restoration of the original retinal neural architecture is beneficial for the amelioration of diseases such as retinitis pigmentosa and AMD. EXAMPLES

[0148] The following examples are not intended to limit the scope of what the inventors regard as their invention, and they are not intended to be all or the only experiments performed.

[0149] Example 1 Restoration and improvement of visual perception is demonstrated in rabbits with ocular blast exposure and retinal damage. Subretinal grafts containing only hESC-3D retinal tissue (no biomaterial / scaffold) are used to treat damaged retinal tissue in rabbits. Structural recovery of tissue and vision is demonstrated using live animal optical coherence tomography (OCT) and after sacrifice using histology and immunohistochemistry. Functional recovery is demonstrated using live animal visual evoked potentials (VEPs).

[0150] Human retinal tissue is generated using clinical grade hPSCs (BIOTIME, INC.). A pilot transplantation experiment in rabbits is conducted to determine the subretinal transplantation procedure for large eye animal models. A blast ocular injury model is generated in rabbits using a shock tube. Then, multiple hESC-3D retinal tissue pieces (~0.1 to ~1 mm in length) are transplanted into the subretinal space of each animal.

[0151] Blast eye injury models can include, for example, those described in Gray, W., Sub-lethal Ocular Trauma (SLOT): Establishing a standardized blast threshold to facilitate diadnostic, early treatment, and recovery studies for blast injuries to the eye and optic nerve. Final report, prepared for: US Army Medical Research and Material Command. Award Number: W81XWH- 12-2-0055, 2015.

[0152] Structural integrity of the retinal tissue will be assessed by OCT and functional integration / visual perception improvement will be assessed by VEP measurements at 1, 2, 3, 4, 5, and 6 months after surgery. Both eyes of each animal will be implanted with retinal tissue and the VEP of each eye will be assessed independently by covering the other eye.

[0153] The following controls can be used: control, one eye (no treatment), control 2, the other eye (sham treatment, i.e., transplanted with only the biomaterial without organoids).

[0154] The implanted hESC-3D retinal tissue grafts are able to synapse on rabbit RGCs and / or second-order retinal neurons, thereby restoring the animals' visual perception (as measured by VEP signals) approximately 4-6 months after surgery. Similar kinetics were observed in a blind rat animal model that received hESC-3D retinal tissue transplants into the subretinal space.

[0155] Cohorts can consist of 8-15 rabbits, so statistical analysis can be performed (one-way ANOVA).

[0156] Example 2 Vision recovery and improvement is demonstrated in rabbits with ocular blast exposure and retinal damage. Subretinal grafts comprising hESC-3D retinal tissue and biodegradable and / or non-biodegradable carriers or scaffolds are used to treat damaged retinal tissue in rabbits. Subretinal grafts can comprise hESC-3D retinal tissue fragments placed on a thin layer of electrospun nanofiber biomaterial scaffolds to form biological retinal patches as described herein. Structural recovery of tissue and vision is demonstrated using live animal optical coherence tomography (OCT) as well as histology and immunohistochemistry after sacrifice. Functional recovery is demonstrated using live animal visual evoked potentials (VEPs).

[0157] Human retinal tissue is generated using clinical grade hPSCs (BIOTIME, INC.). A pilot transplantation experiment in rabbits is conducted to determine the subretinal transplantation procedure for large eye animal models. A blast ocular injury model in rabbits is created using a shock tube. Then, multiple hESC-3D retinal tissue pieces (~0.1 to ~1 mm in length) are transplanted together with a biodegradable carrier or scaffold into the subretinal space of each animal.

[0158] For example, hydrogels (such as those derived from hyaluronic acid, alginate, and others) may be used as biodegradable carriers or scaffolds. Hydrogels can be prepared to gel in situ in the subretinal space from about 1 minute to about 60 minutes after implantation to fixate the implanted retinal fragments in the subretinal space, thereby improving the surgical and functional outcomes. In this study, we demonstrate that implanting hPSC-3D retinal tissue fragments together with biodegradable biomaterials can improve the surgical and functional outcomes of the procedure, resulting in increased VEP signals in more animals 4-6 months after surgery.

[0159] The biological retinal patch or biological retinal prosthetic device consists of several pieces of hPSC-3D retinal tissue placed into a patch of ultrathin biomaterial (approximately 3-5 mm wide and 5-8 mm long) that supports implantation into the subretinal space in rabbits with blast ocular injuries.

[0160] During application, the biological retinal patch may be placed into the retinal space with the retinal tissue positioned to maximize visual recovery. The retinal patch may be applied such that the patch is stabilized within the retinal graft or retinal bleb created prior to application of the patch. The implant may be attached using ancillary materials or procedures.

[0161] Example 3 Using an intraocular injector, hPSC-retinal progenitor cells were delivered into the intraocular space of rabbits (ex vivo experiments). Frozen sections of rabbit eyes receiving transplants of human retinal progenitor cells were stained with anti-human nuclear antibody HNu (red) and pan-nuclear DAPI stain (blue). We demonstrated the presence of human retinal cells (red + blue staining) in the intraocular space of rabbits (blue staining only) delivered using an intraocular injector. Figures 5B to 5D demonstrate that subretinal grafts of human retinal progenitor cells differentiated from human embryonic stem cells (hESCs) can be successfully transplanted into the intraocular space of a large-eyed animal model (rabbit), preserving the retinal layer thickness of the adult mammalian retina for up to 3 months, without detrimental effects on the recipient retina, and without tumor formation. Cells migrate from these grafts and integrate into the recipient retinal layer, reinforcing the recipient retina. Such cells can intermingle with recipient retinal cells in the RGCs and INL and exert paracrine support on surrounding host cells.

[0162] Example 4 Cells from hPSC-3D retinal tissue secrete neurotrophic factors Conditioned medium from hPSC-3D retinal tissue cultures (and undifferentiated hESCs as a control) was assayed for the presence of several key trophic factors, including brain-derived neurotrophic factor (BDNF), glial-derived neurotrophic factor (GDNF), neurotrophin-4 (NT4), nerve growth factor-beta (βNGF), and the pro-survival mitogen basic fibroblast growth factor (bFGF=FGF-2). The concentrations of these neurotrophic factors were read using Luminex technology (RnD Systems), revealing high levels of BDNF and GDNF in the conditioned medium, at least 100- to 1,000-fold above the levels in undifferentiated hESC controls, in addition to bFGF, and neurotrophin concentrations ranging from picograms to nanograms / ml.

[0163] Example 5 Rabbit blast eye injury model A rabbit blast eye injury model was designed based on Jones, K., et al., Low-Level Primary Blast Causes Acute Ocular Trauma in Rabbits. J Neurotrauma, 2016. 33(13): p. 1194-201 to evaluate the potential of the cell preparation described herein to mitigate blast injury-induced retinal degeneration and optic nerve damage, thereby reducing or halting vision loss. Two routes of cell delivery are (i) epiretinal and (ii) subretinal, to explore the route that allows the transplanted cells to have the highest survival rate and most efficient integration into the retina, and to exert the greatest overall therapeutic effect without causing adverse side effects to the host retina. The therapeutic effect of cell transplantation can be evaluated by fundus imaging and OCT (naive retinal morphology), by electroretinography and visual evoked potentials (measures of visual function), and by histopathology of intraocular tissues including retinal grafts after the animals' death (scheduled: 6 months after blast injury). Postmortem analysis of rabbit eyes included histology, fluorescent immunohistochemistry, and confocal microscopy including 3D reconstruction of retinal tissue.

[0164] In this model, a large frame shock tube was used to generate a controllable primary blast wave without secondary or tertiary effects, as shown in Figure 6 (Sherwood, D., et al., Anatomical manifestations of primary blast ocular trauma observed in a postmortem porcine model. Investigative Ophthalmology and Visual Sciences, 2014. 55(2): p. 1124-1132.). The "blast" generated by this shock tube delivered a Friedlander-like waveform with peak static pressures ranging from approximately 7 to 22 Pascals per square inch (psi) (48 to 152 kilopascals, kPa) and a positive pressure peak duration of 3.1 ms. Our data show that a survivable isolated primary blast can produce acute retinal damage in rabbits (levels 2-3 based on the cumulative injury scale (CIS) shown in Table 1).

[0165] (Table 1) Cumulative Damage Scale TIFF2025013961000001.tif36131

[0166] To predict the blast strength that would produce a given CIS injury, a "risk model" was created based on the probability of injury resulting from various blast strengths. Ordinal logistic regression was used to estimate the probability of a given CIS score for each tissue component of the eye, including the retina and optic nerve, for a given level of blast, as shown in Figure 7. A blast with a specific impulse of approximately 725 kPa / millisecond (ms) (approximately 82 psi) is required to produce an 80% probability of CIS 3 retinal injury. Taken together, these data can be used to guide the creation of cohorts of rabbits with relatively uniform severity of retinal injury (but no optic nerve rupture, i.e., animals with "salvageable" vision problems) for statistical evaluation of the impact of cell therapy and retinal progenitor cell transplantation on preserving vision. Although short-range axonal damage in the neural retina can be treated with paracrine trophic factor support, optic nerve disruption (e.g., higher level CIS 3 injuries in shock tubes) results in permanent visual loss that is not reversible with current techniques.

[0167] The model includes approximately 96 specific pathogen free (SPF) graded New Zealand (NZ) pigmented brown rabbits, approximately 5-5.9 pounds each, supplied by RSI Robinson Services, Inc. The rabbits undergo baseline structural and functional assessments, e.g., by fundus imaging, OCT and ERG, VEP recording, prior to undergoing blast ocular injury with a shock tube, and immediately following blast injury. The rabbits are allowed to rest for one day in the ISR animal facility before being transferred to the UTHSCSA animal facility. Retinal organoids are dissociated into single cells, and retinal progenitor cells are transplanted into the rabbit's eye. Approximately four rabbits can be treated per day, with each animal taking approximately two hours to maximize the quality of work.

[0168] We evaluate the survival of human retinal progenitor cells in blast-exposed rabbit retinas, as well as their ability to robustly deliver neuroprotection via paracrine secretion without damaging the host retina. Biomaterials generally promote cell survival in the graft. Epiretinal and subretinal grafts survive in mammalian retinas, although the dynamics of cell integration may differ between rodent and "large eye" models.

[0169] Cells of dissociated hPSC-3D retinal tissue are transplanted into the epiretinal and / or subretinal space of rabbits that have undergone controlled ocular blast injury resulting in damaged retina and / or optic nerve. Neuroprotective effects are then measured by electroretinography (a functional test used to test the photosensitive cells of the eye, the rods and cones of the retina and the ganglion cells that connect them) and visual evoked potentials (a functional test of electrical stimulation of the occipital cortex in response to light outcomes). Histopathological analysis of the ocular tissue may be performed at selected time points after blast injury.

[0170] The effect of subretinal and epiretinal transplants of hPSC-derived retinal progenitor cells with or without supportive biomaterials in mitigating retinal degeneration after blast injury is evaluated in rabbits. Preclinical and clinical studies of stem cells transplanted into the intraocular space have shown therapeutic effects on the degenerating retina. Biomaterials support retinal cell transplants. Subretinal grafts can provide neuroprotection for photoreceptors, while epiretinal grafts can support RGCs. Primary retinal progenitor cells can be structurally and functionally integrated into the host retina.

[0171] The experimental procedure (Methods) may include the following rabbit selection criteria and pilot (P) experiments. Pilot studies of ex vivo rabbit eyes have shown that pigmented eyes are easier to localize the graft. Fl NZ rabbits, approximately 5-5.9 lbs (2.5 kg) and approximately 3 months old, were used to determine the blast intensity that would result in a CIS 2-3 retinal injury resulting in a 50% reduction in ERG amplitude and latency and / or VEP amplitude / latency (as was successful in Dutch Belted rabbits of similar size). Rabbits were prescreened before the blast (to rule out eye disease) and after the blast (to confirm the expected CIS was achieved) by assays such as fundus imaging, OCT, ERG, and VEP. Rabbits will have a CIS 2-3 retinal injury. Grafts will contain approximately 50,000 hPSC-retinal progenitor cells and will be applied to both eyes and to the eyes of three uninjured NZ rabbits. The eyes are then assayed (at +1 day, +1 week, +1 month) e.g., by OCT to demonstrate that the cells have been engrafted. Retinal bulging may be observed. The rabbits can be examined (e.g., IHC) at +1 month after engraftment to determine if the cells have survived. If desired, an immunosuppressive regimen may be used, e.g., prednisone (2 mg / kg topical) + cyclosporine (5.0 mg / animal orally every 12 hours) from day -3 to +8 weeks after surgery.

[0172] Blast ocular injury: A shock tube (described above) is used to produce a CIS 2-3 retinal injury in rabbits (Table 1). Imaging (fundus photography, OCT) and electrophysiology (ERG, VEP) can be performed 1 day before and 2 days after the blast, as shown in Table 2.

[0173] Intraocular implantation tools: The grafts can be applied using any suitable implantation tool. For example, a system of a Micro-4 controller, a 100 μl Hamilton syringe, and a World Precision UMP-3 pump connected to a microcapillary (1.0 mm outer diameter with pulled polished opening) for intraocular cell delivery can be used. Histology, fluorescent immunohistochemistry, and confocal immunofluorescence microscopy can be performed on lightly fixed frozen sections of the eye.

[0174] For example, about 50,000 human retinal progenitor cells dissociated from hPSC-derived retinal tissue (organoids) using about 40-50 microliters of papain (Nasonkin, I., et al., Long-term, stable differentiation of human embryonic stem cell-derived neural precursors grafted into the adult mammalian neostriatum. Stem Cells, 2009. 27(10): p. 2414-26) can be used for grafting. If the cells are transplanted with a carrier or scaffold, such as the hydrogel-like HYSTEM® biomaterial (gel), the cells may be premixed with the carrier or scaffold before each transplant. We transplant heat-activated (dead) retinal progenitor cells (with or without carrier or scaffold) into the "control" (other) eye as shown in Table 2.

[0175] Table 2. Study design of cells and bioprosthetic patch (cells + bioprosthetic material) TIFF2025013961000002.tif36151

[0176] Initial analysis is performed by evaluating the eye in vivo 1 day before the blast and 2 days after the blast (e.g., OCT = retinal thickness, presence of graft, ERG, VEP - visual function test). Cells are then transplanted and measurements are taken periodically (Table 2). We expect that at +1 day after the blast, animals will have at least a 50% reduction in ERG and VEP amplitude and / or latency compared to their baseline levels. The baseline level of functional recovery is an increase in electrophysiological response to at least about 25%, 30%, 40%, 50%, 60%, 70%, or 75% of baseline. Once animals reach this level of recovery or have not recovered at +6 months after blast exposure, they will be euthanized. Eyes will be enucleated and optic nerves harvested for frozen IHC analysis to delineate the effect of the graft on retinal preservation. Cell survival, graft retention, integration of human cells into the rabbit retina, changes in retinal thickness, levels of glial and fibrous scar formation, retinal remodeling, cell death, and retinal structure will be measured 6 months after surgery. The experiment will be partially blinded. Rabbits will be assigned ID numbers. Laboratory technicians will not know which eye of each rabbit received live cells until the end of the experiment. This will allow for the most objective determination of neuroprotective efficacy. Laboratory technicians will not know the rabbit IDs when performing histology and IHC analysis until the end of the experiment.

[0177] Power analysis, statistical evaluation, sample size, and controls: According to Okuno et al., the variability (relative standard deviation [RSD], or coefficient of variation) of VEP amplitude was about 12%, whereas the latency was not (RSD about 3%). Thus, VEP is a robust measure of visual function. Using Okuno's formula as the basis for power calculation, we estimated that the minimum sample size required was 7 for 80% power (l-β, where β is the probability of type II error) and sufficient statistical power to detect a difference in the means with a p-value of 0.05. The sample size could be 10 eyes / cohort, which is sufficient for statistical evaluation of visual function changes (VEP) by ANOVA method, allowing for some group reduction (e.g., due to graft failure).

[0178] Immunosuppressants can be used to increase cell viability. Retinal thickness and VEP are not significantly affected. Also, carriers such as hydrogels (e.g., HYSTEM® biomaterials) (BioTime, Inc.) containing trophic factors (e.g., BDNF embedded in the gel for sustained release) can be used to increase the effect on retinal thickness and VEP.

[0179] The specific cell dose transplanted into the adult CNS allows for robust integration of the cells. Although drug-based therapy prospects (dose-response relationship) are important, one aspect of this work is to find a cell dose that does not have deleterious effects on the recipient retina (e.g., leaving a bulge of unintegrated cells in the subretinal space or growing an epiretinal membrane in the epiretinal space).

[0180] Experimental Procedure (Methods): Doses of 10,000, 100,000, and 250,000 cells are tested for the creation of grafts integrated into the rabbit retina. In this case, the selection of the three cell doses may be centered around 50,000 (e.g., 30,000; 45,000; 65,000 cells / graft). The experimental design is shown in Table 3. Ten rabbits can be assigned to each dose level.

[0181] Table 3. Study design to optimize subretinal vs. epiretinal cell dose with or without carrier / scaffold. TIFF2025013961000003.tif44153

[0182] Each animal will have one eye grafted and the other eye implanted with dead cells. The route of application (subretinal or epiretinal, with or without biomaterial) will be chosen based on initial results.

[0183] Paracrine factors resulting from the graft that provide the best neuroprotection can be identified, and these molecules can be overexpressed by the graft or / and embedded in the supportive biomaterial.

[0184] Provided herein is the determination of the time to deliver retinal cell therapy following retinal blast injury to mitigate vision loss in a rabbit model.

[0185] Retinal cells begin to die immediately after blast injury. RGCs and photoreceptors are most susceptible to cell death. However, decreased vision during the first few days after blast ocular injury does not necessarily mean vision will be lost; this will only become evident after approximately 3-4 weeks. As cell death progresses, vision will gradually decrease. During this time, at least some vision may be salvageable. Delayed analysis (up to +2 weeks after blast injury) will determine whether therapeutic interventions can still protect the retina. Results will be meaningful for developing vision preservation approaches for wounded soldiers during triage.

[0186] Cell preparation, transplantation, randomization to reduce bias, cohort size, sampling, manipulation, and power analysis are described above. In addition to the study design outlined in Table 4 and measurements of retinal thickness and retinal cell preservation (as described), analyses of comparison and quantification of cell death in graft-treated rabbit retinas at +3 days vs. +2 weeks post-blast are performed. Cleaved Caspase-3, γH2AX (early marker of apoptosis), and Tunnel staining (late marker of cell death) can be used. As a second readout, the presence of activated microglia (Iba-1 marker) as a measure of retinal remodeling and quantification of inflammation in control and experimental cohorts can be performed. Differences in synaptic bouton preservation between the inner and outer plexiform layers can also be determined.

[0187] Table 4. Study design to test the effect of a 2-week delay in retinal cell transplantation after blast on retina and vision preservation TIFF2025013961000004.tif44151

[0188] Cell therapy may be planned such that earlier treated retinas (+3 days post-blast) will have improved preservation of retinal thickness, lower levels of apoptosis and retinal remodeling, and better preservation of the synaptic layer.

[0189] Example 6 Wild-type cat eyes were subjected to pars plana vitrectomy and then transplanted with hPSC-3D retinal tissue into the subretinal space (n=3 eyes). hPSC-3D retinal tissue can be transplanted by any suitable method, for example as described in Seiler, MJ, et al., Functional and structural assessment of retinal sheet allograft transplantation in feline hereditary retinal degeneration. Vet Opthalmol, 2009. 12(3): p. 158-69, which is incorporated herein by reference in its entirety. Eyes were clinically examined by fundus examination and spectral domain optical coherence tomography (OCT) for adverse effects due to the presence of the subretinal graft. Cats were euthanized 5 weeks after transplantation and immunohistochemistry of retinal sections was performed using human-specific antibodies (HNu, Ku80, and SC121) to determine the location, differentiation, and layering of the graft in the subretinal space. Anti-inflammatory doses of oral prednisone were administered throughout the study.

[0190] Fundus examination did not reveal any gross retinal inflammation. OCT imaging 3 weeks after transplantation showed that the grafts were in the correct location in the subretinal space, as shown in Figure 8. Immunostaining of retinal cryosections with HNu and Ku80 antibodies also revealed the presence of human-derived retinal tissue grafts in the cat subretinal space, as shown in Figure 9. The majority of cells in the grafts had cytoplasmic staining, but not nuclear staining. These results demonstrate that hESC-derived retinal tissue can be successfully transplanted into the cat subretinal space without severe inflammatory reactions.

[0191] Example 7 To demonstrate that implanted human embryonic stem cell-derived 3D retinal tissue (hESC-3D retinal tissue) has the ability to produce lamination within the graft, hESC-3D retinal tissue was delivered subretinally to blind immunodeficient rats, SD-Foxnl Tg(S334ter) 3 Lav(RDnude) rats for treatment. Figure 10A shows an image of hESC-3D retinal tissue (retinal organoid) excised from a dish prior to transplantation. Figure 10B shows an image of excised retinal organoid growing in a dish prior to transplantation. Figure 10C shows an additional image of retinal organoid growing in a dish. After implantation and rat euthanasia, histological analysis was performed on the subretinal space 10 weeks after implantation. Figures 10D and 10E show lamination of the graft. Figure 10F shows outer segment-like processes in the outer layer immediately adjacent to the rat RPE.

[0192] Example 8 We demonstrated that hESC-3D retinal tissue could be transported overnight in two different conditions (low temperature in Hibernate-E medium and with or without BDNF in the original medium at 37°C) without affecting the viability of the retinal tissue. Upon arrival, the tissue was fixed and IHC was performed with Cleaved Caspase-3 (an apoptosis marker) showing positive cells (arrows in Figure 11), indicating that the retinal tissue maintained viability after overnight transport in Hib-E at 4°C.

[0193] The feasibility of inducing 3D human retinal tissue bearing all retinal layers (PR, second order neurons, retinal ganglion cells) and RPE from hESCs has been demonstrated (see, e.g., International Publication WO2017 / 176810, incorporated herein by reference in its entirety), and electrophysiology has demonstrated that increased synapse formation occurs concomitantly with increased electrical activity in hESC-3D retinal tissue.

[0194] In 6- to 8-week-old hESC-3D retinal tissues, only a portion of neurons express Na + and K. +In 12- to 15-week-old hESC-3D retinal tissue aggregates, nearly all retinal neurons tested were easily excited and showed robust Na currents. + and K. + The current was shown.

[0195] Example 9 A 1.52 mm outer diameter (OD), 1.12 mm inner diameter (ID) microcapillary from World Precision Instrument can be used. Immunosuppression regimen of systemic cyclosporine from day 7 onwards prior to transplantation, techniques for delivering hESC-3D retinal tissue to the subretinal space in cats and imaging methods (e.g., Spectral OCT, RetCam at several different time points including immediately after transplantation and immediately prior to killing the animals) can also be used to deliver viable hESC-3D retinal tissue to the subretinal or epiretinal spaces of large eyed animals.

[0196] 12A-12C show the surgical team transplanting hESC-3D retinal tissue into the subretinal space of a wild type cat. FIG. 12D shows the intraocular pressure adjustment device and the RetCam device imaging the graft. FIG. 12E shows two ports inserted into the cat's eye for intraocular surgery. FIG. 12F shows the retinal detachment (bleb) for transplanting hESC-3D retinal tissue into the subretinal space. FIG. 12G shows the cannula for injecting hESC-3D retinal tissue. FIG. 12H shows hESC-3D retinal tissue in the subretinal space of a wild type cat imaged with RetCam. FIG. 12J shows a cross-sectional OCT image of hESC-3D retinal tissue 5 weeks post-transplantation placed in the subretinal space of a wild type cat. FIG. 12K shows a 3D reconstruction of the OCT image to estimate the overall dimensions of the graft.

[0197] Example 10 Immunohistochemical analysis of hESC-3D retinal tissue grafts 5 weeks after transplantation into the subretinal space in wild-type cat eyes demonstrated tumor-free structural and synaptic integration of the hESC-3D retinal tissue into the retina of large-eyed animals. Preservation of cat eyecups with grafts for cryohistology / IHC, confocal IHC with retinal-, human-, and synapse-specific antibodies was successfully performed. Figure 13A shows a cat eyeball with subretinal grafts that was PFA-fixed, cryoprotected, and OCT-saturated in preparation for sectioning. Figure 13B shows a cross-section of a frozen cat eyeball in OCT. Figure 13C shows a 16 μ thick section of a cat eyeball in OCT, showing the graft as a central retinal bulge. Figure 13D shows a magnified image of a region of the frozen section showing preservation of the hESC-3D retinal tissue graft.

[0198] FIG. 13E shows IHC of cat retinal sections with hESC-3D retinal tissue grafts 5 weeks after transplantation into the subretinal space. The grafts show the presence of numerous CALB2 (calretinin) positive neurons, with the arrow pointing to CALB2[+] axons connecting the human graft to the cat's ONL. FIG. 13F-H show hESC-3D retinal tissue grafts in the cat's subretinal space stained with HNu, Ku80, and SC121 human (but not cat) specific antibodies, respectively. These results demonstrate that indeed the human tissue has been transplanted into the correct location in the cat's subretinal space. FIG. 13I shows staining with BRN3A (a marker for RGCs) and a human nuclear marker. The asterisk indicates the area with the markers in the main image and is magnified in the inset. These results indicate that the cells within the grafts are maturing into RGCs. Figure 13J-K show staining with antibodies specific for human (but not feline) synaptophysin (hSYP) and the axonal marker NFL (specific for both feline and human neurons) and show the presence of punctate staining (arrows), which indicates possible synapses formed by human neurons integrating with feline neurons. Human punctate staining was observed at the border of the feline ONL and the hESC-3D retinal tissue graft, indicating possible initiation of synaptic connections. The distribution pattern of punctate staining (red) also demonstrates that developing human synapses are connecting with the recipient retina.

[0199] Immunohistochemical (IHC) evidence of connectivity with hESC-3D retinal tissue grafts in the subretinal space of wild-type cats was demonstrated 5 weeks after transplantation. Figures 14A and 14B show the human (but not cat) specific synaptophysin antibody hSYP (red) and calretinin (green), which stains both cat and human neurons. hSYP stains human puncta in the cat ONL (arrows). Figures 14C and 14D show lower magnification images and provide a schematic of a large piece of cat retina with hESC-3D retinal tissue grafts. hSYP staining originates from the graft, stains the graft and parts of the ONL facing the graft, but does not stain the cat retina adjacent to the graft.

[0200] Figures 15A-C show calretinin[+] axons (arrows) connecting the feline INL with calretinin[+] human cells in the graft. Under higher magnification, these axons were found to extend from the feline cells into the human graft and from the human calretinin[+] cells into the feline INL. Figures 15D and 15E show mature-appearing calretinin[+] neurons in the graft and calretinin[+] axons found throughout the graft.

[0201] Figures 16A-E show staining of the hESC-3D retinal tissue graft edge in the cat subretinal space. SC121 human cytoplasmic specific antibody (red) and Ku80 human nuclear specific antibody (green) stain the human retinal graft but not the cat retina. The images show connectivity between the graft and the host. Figure 16D shows axons from the hESC-3D retinal tissue graft wrapping around the cat PR in the layer immediately adjacent to the graft (arrows), and some SC121+ human axons can be seen crossing over the cat ONL (arrows in Figures 16B, FIG, 16E).

[0202] These results indicate that the distribution pattern of staining is indicative of tumor-free survival and maturation of the grafted cells as well as synaptophysin-stained synaptic connectivity resulting from the graft. No tumors developed in any of the feline subjects.

[0203] Example 11 We further demonstrate the histology and IHC-based synaptic connectivity mechanisms, and the functional determination at the electrophysiological level, of hESC-3D retinal tissue transplanted into the degenerating retina in at least two large-eyed genetic RD animal models. We demonstrate that hESC-3D retinal tissue at specific developmental time points of differentiation can structurally and synaptically integrate with the degenerating recipient retina and serve as a "biological patch" to restore vision in subjects with retinal degeneration, retinal disorders, and diseases, including progressive retinal degeneration. Furthermore, demonstrating the positive therapeutic impact of hESC-3D retinal tissue transplantation into large-eyed animal models with retinal degeneration allows for further enhancement of the bioprosthetic retina consisting of multiple hESC-3D retinal tissue pieces on the bioprosthetic material. We further demonstrate the positive therapeutic effects of hESC-3D retinal tissue transplantation into two large-eyed animal models with retinal degeneration (Pde6a[- / -] dogs and Aipl1 - / - cats), and, if necessary, two additional large-eyed animal models (Cngb1 - / - Dogs and Crx + / - Cats) can be used.

[0204] Full-field ERG and mfERG are performed to assess the function of the retina during degeneration and to compare the changes in retinal function in the perigraft (central retina) and periphery of subjects with grafts and in control subjects. Retinas with PR degeneration, especially in the sites above the graft, are assayed for electrical activity of individual RGC cells using established MEA techniques. Multi-electrode arrays allow for the reading of many individual RGCs at once, thus avoiding the need to use laborious patch clamp techniques on individual RGCs. Patch clamp techniques are less informative and may not indicate RGCs that have synaptic connectivity with the hESC-3D retinal tissue graft. Recordings can be performed for 1-2 hours in an oxygen chamber where retinal viability is maintained, thus allowing accurate readings. These assays allow for the analysis of correlations between synaptic connectivity and structural (histology / IHC using human synaptophysin, human SC121 antibody, and WGA-HRP transsynaptic tracer) and functional (electrophysiological) levels of individual retinal cells. mfERG allows precise localization of the activity of the host retina (vs. individual cells) surrounding the graft. Multielectrode arrays allow demonstration that the graft acts via cell replacement rather than (or in addition to) via neuroprotective mechanisms / cell fusion.

[0205] Since MEA recordings take approximately 1-2 h and involve gradual deterioration of the retinal structure, hESC-3D retinal tissue may be transplanted into both eyes of Pde6a dogs and Aipl-1 cats (6 animals = 12 eyes / each model), allowing one eye to be dedicated to multielectrode array readout and the other eye to be used for histology / IHC readout.

[0206] We assay hESC-3D retinal tissue expressing the transsynaptic tracer, wheat germ agglutinin-horseradish peroxidase (WGA-HRP), in vitro and in vivo, and analyze transplants of hESC-3D retinal tissue in both canine (Pde6a[- / -]) and feline (Aipl-1[- / -] RD models, six of each) to evaluate the ability of both models to maintain grafts and promote synaptic integration. Histology, IHC, and xenograft-specific antibodies can also be used. In vitro electrophysiology (MEA), along with high-resolution histology, immunohistochemistry, mfERG, and VEP, can be used to evaluate transplantation outcomes.

[0207] Herein, we provide methods to determine the mechanism of synaptic connectivity between grafts transplanted into three cohorts of animals (at RD onset, partially degenerating retinas with approximately 50% preserved ONL thickness, and retinas with mostly / completely degenerated PR) and the recipient degenerating retina. Using both in vitro and in vivo electrophysiological methods, as well as visually guided behavioral tests, we can delineate the degree of visual recovery in visually impaired subjects.

[0208] Transplantation of bioprosthetic retinas (grafts larger in size than individual hESC-3D retinal tissue constructs or organoids) is also performed. Bioprosthetic retinas are composed of multiple hESC-3D retinal tissue pieces placed on a bioprosthetic material or carrier or scaffold (e.g., hydrogel-based, e.g., HYSTEM®) that carries thousands of PRs (=biological pixels) and allows for the restoration of visually guided behavior. This bioprosthetic retina can also be customized to treat specific retinal diseases or disorders, e.g., macular degeneration, and the patch can be redesigned to carry primarily cones, allowing the reconstruction of a macula that is mostly composed of cones.

[0209] Wheat germ agglutinin-horseradish peroxidase complex (WGA-HRP) can be used as a transsynaptic tracer. 3D retinal tissue can be derived from tracer[+] and tracer[-] hESCs, which can be co-cultured for 2-3 months and tested for HRP (using the HRP substrate DAB) in the tracer[-] retinal tissue, which shows migration of the transsynaptic tracer from the tracer[+] retinal tissue. Tracer[+] human retinal tissue can be co-cultured with dog and / or cat fetal retina for 2-3 months and tested for either (1) WGA-HRP migration or (2) chimeric human / non-human synapse formation to determine synaptic connectivity. Although the feasibility of the latter method using antibodies specific for human synaptophysin (hSYP) and human cytoplasm (SC121) has been demonstrated, we will also try WGA-HRP as it is more sensitive in detecting chimeric (human-non-human) synapses. The tracer[+] retinal tissue constructs can then be implanted into the subretinal space of young (4-5 weeks) Pde6a- / - dogs and six Aipl-1- / - cats (receiving grafts in both eyes). Animals can be imaged using RetCam and optical coherence tomography (OCT), animals can be sacrificed at, for example, 6 months, samples stained with DAB, hSYP, and SC121 to determine graft / host synaptic connectivity (one eye / animal), and the other eye can be electrophysiologically tested ex vivo with a multielectrode array (MEA).

[0210] We can demonstrate both synaptic integration (by transsynaptic tracers and IHC), outgrowth of the outer segments of PRs in the graft, and further demonstrate functional integration (finding RGC activity by MEA around / above the graft site) by 6 months post-graft. A neuroprotective effect with young hESC-derived retinal organoid grafts can also be demonstrated. Observation of MEA signal may indicate that the slowed progression of regeneration or retinal degeneration (compared to the retina 3-4 mm outer from the graft) is due to specific PR replacement mechanisms, not just neuroprotection.

[0211] In one aspect, we engineered hESCs expressing wheat germ agglutinin-HRP gene tracer under the control of the elongation factor-1 alpha (EF-1α) promoter, scaled up the induction of hESC-3D retinal tissue to produce, identified hESCs (DNA fingerprinting), performed karyotype analysis, and implanted the engineered hESC-3D retinal tissue into 6 Pde6a- / - dogs and 6 Aipl-1- / - cats (both eyes) and analyzed by OCT, whole eye ERG, mfERG, MEA, and VEGF. P (using control Pde6a- / - dogs and control Aipl-1- / - cats as control readouts for retinal degeneration), wait 6 months, sacrifice the animals, remove the eyes and retina with grafts, characterize changes in RD retinal function at the site on the graft (using patch clamp techniques on individual RGCs and also MEAs), then fix the tissue with grafts and characterize synaptic connectivity between the graft and recipient and maturation of the transplanted hESC-3D retinal tissue using antibodies against retina-specific immunophenotypes.

[0212] hESC-3D retinal tissue can be derived using cGMP grade hESC. Differentiation kinetics can be determined in several different lines of cGMP-hESC from companies such as, for example, ES Cell International Pte. Ltd. The cells from ES Cell International Pte. Ltd. have normal karyotype and are fully characterized.

[0213] Synaptic connectivity within and between the hESC-3D retinal tissue and the degenerating recipient retina can be exploited to create functional biological "retinal patches" that allow visual information to be transmitted and received from the PRs of the graft to the RGCs of the recipient retina. Rapid degeneration of the recipient retina can bring the graft and RGCs in close proximity, facilitating graft-host connectivity. Converging evidence suggests that 6-12 week old hESC-3D retinal tissue can engraft, differentiate, laminate, and synapse with the recipient retina in dogs and cats with RD. hESC-3D retinal tissue has an RPE layer, which favors survival, maturation, and outer segment development of PRs during grafting.

[0214] WGA-HRP transsynaptic tracer can be used to demonstrate synaptic connectivity between grafts and hosts. WGA-HRP is expressed from a strong ubiquitous promoter, EF1alpha, and can be engineered by introducing an EF1alpha-WGA-HRP construct in a custom-made lentiviral vector (e.g., GeneCopoeia) into hESCs (from which hESC-3D retinal tissue is derived) and allowing cross-breeding of human / dog or human / cat synapses once synaptic connectivity is established at 6 months. hESC-3D retinal tissue has been shown to (i) initiate synapse and axon formation at approximately the 8th week of development and (ii) show signs of punctate synapses between grafts and recipient wild-type cat retina within 2 months of transplantation. High-resolution confocal immunohistochemistry can be used with hSYN antibody (specific for human presynaptic parts but not cat / dog synapses) and HNu (human nuclei) antibody to demonstrate human synapses around recipient retinal neurons. We can look for hSYN[+] terminal boutons on recipient neurons that do not have HNu[+] nuclei, and separately, we can look for SC121[-] axons that have hSYN[+] terminal boutons.

[0215] As an additional control, animals with retinal degeneration mutations that have not been surgically manipulated can be isolated and the same retinal sites examined by MEA, and the results can be compared to where the grafts were placed, not far from our "landmark" optic nerve.

[0216] For ex vivo electrophysiology experiments, a multi-electrode array (MEA) can be performed on the fellow eye. After MEA, the integrity of the retinal tissue is gradually lost, so this tissue cannot be used for histology. Therefore, MEA readings can be performed on samples from approximately 6 dog and 6 cat grafted eyes (attempting in vivo mfERG and VEP before sacrificing the animals), while histology and IHC data can be generated from the fellow eye (also 6 dog and 6 cat grafted eyes). mfERG can be performed on both eyes of each animal before sacrificing the animals, and the signals from the perigrafted retina can be compared with the signals from the fully degenerated retina and the PR (negative control) far removed from the graft.

[0217] hPSCs can be cultured on Laminin-521 or growth factor reduced (GFR) MATRIGEL or vitronectin using mTeSR1 medium. Custom made lentiviral vector transduction synaptic reporters (by companies such as Genocopoeia) can be introduced into hESCs, isolated using drug selection puromycin in 10 μM Rho-kinase inhibitor (ROCK) for 2 weeks, and colonies can be assayed for WGA-HRP expression, expanded, and stored in liquid nitrogen. Derivation of hESC-3D retinas can be performed according to the methods described herein. Eyeballs can be removed immediately after animal sacrifice (MSU protocol or other protocols), immersed in ice-cold fresh 4% paraformaldehyde / PBS pH 7.6-8.0, the anterior chambers dissected out, and the eyecups fixed for an additional 15 min at 4 °C for histology, IHC, and pre-embedding methods. Eye cups can be cryopreserved in 20%-30% sucrose and flash frozen in OCT / sucrose. Optic nerve and brain tissue from each animal can also be preserved (to trace HRP[+] axons to determine if WGA-HRP is transported from the graft through the recipient RGCs to the superior colliculus via RGC axons). For analysis of human grafts and human / rat and human / cat synapses, selected sections containing hESC-3D retinal tissue grafts can be stained with human-specific HNu and α-synaptophysin antibodies. IHC can be performed using the antibodies / protocol described in Singh, RK, et al., Characterization of Three-Dimensional Retinal Tissue Derived from Human Embryonic Stem Cells in Adherent Monolayer Cultures. Stem Cells Dev, 2015. 24(23): p. 2778-95, or using an alternative protocol.

[0218] hESC-3D retinal tissue grafts can be transplanted into three cohorts of cyclosporine-immunosuppressed animals: (i) before the onset of retinal degeneration, (ii) when one or two photoreceptor layers are still present, and (iii) after the progression of degeneration. Retinal tissue grafts can be derived from canine induced pluripotent stem cells (iPSCs), and whether immunocompatibility with canine recipients enhances the engraftment and functional integration of bioprosthetic retinal grafts can be assessed. Grafts may be monitored for 12 months using RetCam and OCT. In addition, functional assays such as electroretinography (ERG), multielectrode array (MEA) recordings, visual evoked potentials (VEPs), pupillary light reflex, and visually guided maze navigation may be used to test retinal photosensitivity and visual function at 3, 6, 9, and 12 months. Animals can be sacrificed at 12 months after transplantation to determine synaptic integration.

[0219] The mechanism of synaptic connectivity between the graft and the recipient degenerating retina can be determined by performing the transplantation procedure described herein on partially degenerating retinas with 50% preserved ONL thickness and / or retinas with mostly / completely degenerated PR at the time of RD onset in animals. Both in vitro and in vivo electrophysiological methods, as well as visually guided behavioral tests, can be used to delineate the degree of visual recovery in blind animals.

[0220] Spectral domain-OCT and RetCam imaging can be performed 2 weeks after surgery, followed by high-resolution spectral domain (SD)-OCT at 3 weeks, and additional SD-OCT scans up to 1 year later (at 2, 3, 6, 9, and 12 months after implantation) to select "good" grafts (one example of criteria may include large grafts in the central retina). Animals with excessive surgical trauma / ocular hemorrhage may be excluded at the initial RetCam and SD-OCT scans. Optokinetic testing can be performed on implanted and sham-operated cats every 2 months, starting 1-2 months after surgery. This test evaluates whether the cat can see moving strips of a certain thickness (cycles / degrees) and determine the spatial threshold. Each test can be performed twice, on the same or the next day. Videos can be evaluated by two independent investigators blinded to the condition of the animals. Due to the heterogeneity of the study, group sizes can be a minimum of 6 animals.

[0221] Multifocal (mf)ERG is a method that allows comparison of PR function between different parts of the animal's retina and allows pinpointing of fine electrophysiological differences between the graft site and the host retina with degenerated PRs around the graft. Pupillary light reflex can be performed and pupillometry recorded for all animals and sham surgery at approximately +2 and +3 weeks after surgery, and then at approximately 2, 3, 6, 9, and 12 months after transplantation, up to approximately 1 year. VEP recordings can be performed for all animals and sham surgery at +2 and +4, 6, 9, and 12 months after surgery, up to 1 year.

[0222] For histology / IHC, the eyes can be removed (immediately after killing the animals), fixed in ice-cold 4% paraformaldehyde (PFA) for 2 hours, then washed three times in ice-cold PBS (~30 min each), cryoprotected with sucrose (final concentration ~30% in PBS), and the graft-bearing eyes (e.g. selected by SD-OCT) can be sectioned on a Cryostat to produce 12 μm frozen sections. Histology can be performed on every 20th section using hematoxylin and eosin (HE) or crestal violet (CV) to identify the sections bearing the graft. IHC can be performed using antibodies specific for human (but not cat / dog) tissues (SC-121, Ku-80, or HNu, NF-70), antibodies specific for various cat / dog human specific retinal cell types (rods and cones, PR, bipolar cells (e.g., CaBP5, PKCα, SCGN), amacrine (e.g., calretinin), RGC markers (e.g., BRN3A, BRN3B), and synapses (SYP, SYT, BSN, PCLO, CTBP2, mGluR6, PSD-95, etc.), such as the human synaptophysin specific hSYP antibody, but not cat / dog.

[0223] MEA recordings can be performed by removing the eye immediately after killing the animal and transferring it to an oxygen chamber where the retinal piece with the graft can be carefully isolated and left there throughout the recording procedure. For immuno-EM, the following steps can be taken: immediately after removal, the eye is fixed in about 3% glutaraldehyde and about 2% PFA, washed, embedded in a gelatin-albumin mixture and hardened with glutaraldehyde, sectioned on a vibratome, IHC with hSYP antibody by non-fluorescent method (secondary antibody is horseradish peroxidase), resin embedding, and resectioning at ultrathin level.

[0224] Morphological and functional evaluation of bioprosthetic retinal grafts Several morphological and functional assessments can be performed to determine the quality of the transplantation procedure and whether the graft induces photosensitivity in the degenerated retina. Fundus imaging and optical coherence tomography can be performed periodically after transplantation to monitor the appearance of the graft and the condition of the retina. To investigate graft-induced photosensitivity, various behavioral and electrophysiological tests can be performed immediately before transplantation and at 3, 6, 9, and 12 months after transplantation, including (1) visually guided behavior; (2) in vivo imaging of the pupillary light reflex; (3) in vivo electroretinography to determine retinal light response; (4) recording of visual evoked potentials to determine the transmission of retinal light response to the visual cortex; and (5) in vitro multielectrode array recording to determine the light response of ganglion cells in the transplanted retinal region.

[0225] Wide-field color fundus imaging can be performed using a fundus video camera (e.g., RetCam II, Clarity Medical) to document the placement of the graft immediately after implantation and periodically, thereby allowing the appearance of the graft to be monitored and any inflammatory reactions to be noted. Monitoring can be performed in conscious animals after pupil dilation (tropicamide) and application of local anesthesia (proparacaine).

[0226] Spectral domain-optical coherence tomography (OCT). Scanning laser ophthalmoscopy (cSLO) and retinal cross-sectional imaging (OCT) of the graft can be recorded using a Spectralis instrument (e.g., from Heidelberg Engineering). This is performed under general anesthesia (e.g., induced with propofol, intubated, and maintained with inhaled isoflurane in oxygen) with the animal on a heating pad and maintained at 37°C. The eye can be maintained in primary position with a lid retractor and conjunctival support sutures. Both infrared and autofluorescent cSLO imaging can be performed. The appearance of the graft and host retina can be recorded with high-resolution line and volume scans, and enhanced depth imaging (EDI) protocols can be used if necessary. Repeated imaging can be performed and aligned with previous images using Heidelberg eye tracking software. In this way, retinal morphology and retinal layer thickness of both the graft and the overlying host retina can be determined. It provides morphological data on the condition of the retina and associated abnormalities such as retinal detachment, edema, or thinning of the retina itself that may occur after the implantation procedure. Figure 17 shows a RetCam image of a retinal tissue bioprosthesis implanted in a cat, taken immediately after implantation into the subretinal space.

[0227] Functional Determination Protocol Vision testing of dogs can be performed using a four-choice visual testing device that has been used in the past in retinal therapy experiments. The measures are correct exit choice and exit time, providing an objective assessment of vision at scotopic, mesopic and photopic lighting levels. The test can identify rod as well as cone mediated vision. One eye can be occluded with an opaque contact lens and tested one at a time. Vision testing of cats can consist of several different techniques. These include the assessment of the optokinetic reflex (OKR) using a custom optokinetic device, and platform identification. The OKR test is a technique for assessing the vision of cats. The cat's behavior of chasing a moving object can also be exploited, i.e. laser pointer tracking. Finally, techniques can be used to assess the visual acuity of cats, for example their ability to jump to a platform indicated by a visual cue. In this technique, cats are trained to be rewarded for identifying the indicated platform and receive negative reinforcement for choosing the incorrect platform.

[0228] In vivo pupillary light reflex (PLR) imaging can be used to determine whether the graft enhances retinal photosensitivity. PLR is almost entirely mediated by native photosensitive retinal ganglion cells (ipRGCs), but it is useful for determining the function of ipRGCs as well as rod / cone circuits. This is because the light response of ipRGCs is both direct through their photosensitive pigment melanopsin and indirect through synaptic inputs from rods and cones. PLR can be measured at a total of five time points as previously described. At each time point, PLR imaging can be performed one day before in vivo ERG recordings are obtained in the same animals. To minimize diurnal variation, all PLR imaging can be performed at approximately the same time of day.

[0229] The evening before each PLR imaging, animals are allowed to dark adapt overnight. The next morning, animals are anesthetized under dim red light. After turning off the red light and allowing animals to dark adapt for 10 minutes, the grafts are located in the transplanted eye using the RETImap system (Roland Consult). This device is based on confocal laser scanning technology, which uses an infrared laser to scan the retina without light adapting the retina or producing a visual response, and acquires images of the fundus by cSLO to identify the transplanted area. The system can then be used to generate visible wavelengths of light that focally illuminate the graft-containing area in the transplanted eye, and an eye tracker (e.g., SR Research EyeLink 1000 Plus) can be used to image the non-transplanted eye under infrared illumination to look for congruent PLR. Four different intensities can be presented, spanning at least 3 log units. As a control, focal illumination can be delivered to the corresponding area in the non-transplanted eye, and the other eye is imaged for congruent PLR. Images of the pupil captured by the eye tracker can be transmitted in real time via a frame grabber to a separate computer for offline analysis of pupil diameter. This measurement can utilize LabVIEW-based image processing routines. In cats, the horizontal diameter of the mid-pupil can be measured. The peak pupillary constriction can be measured at each recording. For each stimulation intensity, the peak constriction due to illumination of the transplanted eye can be compared to the peak constriction due to illumination of the non-transplanted eye using a Mann-Whitney U test. If the graft is truly capable of enhancing photosensitivity, it is predicted that light stimulation of the transplanted eye will result in a stronger pupil constriction than light stimulation of the non-transplanted eye.

[0230] In vivo electrophysiology can determine the ability of transplanted hESC-derived retinal tissue bioprosthetic implants to support light-induced activity of the recipient retina. A range of in vivo electrophysiological assessments can be used. ERG techniques can indicate whether the graft is functional and whether it improves retinal function. VEPs can indicate whether there is transmission to the visual cortex and, together with visual and PLR testing, can determine the overall feasibility of the transplant technique to improve retinal function. These measurements can be performed in intact animals and can be repeated over long follow-up periods. In animal models with genetic or artificial retinal degeneration, full-field or focal flash-evoked ERGs can determine the status of retinal function. After transplantation of stem cell-derived suspension or sheet implants, focal stimulation of the graft may be more effective than full-field stimulation of the graft, especially if the host retina is degenerated. ffERGs can also be performed. Focal and multifocal ERG testing can be performed using the RETImap system. Identification of the transplanted area can be performed using the RETImap described above for PLR imaging, which can also be used to focus light stimuli on the area to elicit focal ERGs. Each transplanted area can be stimulated and the response recorded and compared to both non-transplanted retinal areas and the same area in a control (untreated) eye. Alternatively, multifocal ERGs can be performed.

[0231] If the graft successfully forms photoreceptors and synaptic connections with the host retina, thereby providing light-activated neural activity, transmission of visual information from the optic tract to the central nervous system can be achieved. To demonstrate this, recordings from the visual cortex (corresponding to human eye site 17) can be performed. This can be done simultaneously with ERG recordings by attaching cutaneous or subcutaneous electrodes to the occipital region of the animal. Assuming functional integration of the graft has occurred, VEPs can also be elicited by the same stimuli that can be used to generate ERG responses. Flash stimuli (no pattern) and pattern stimuli (checkerboard or grid) can be used, which can be created by the RETImap system.

[0232] Animals can be dark adapted overnight and prepared for recording under dim red light. Anesthesia, pupil dilation, and eye positioning can be used as described herein for OCT. First, a dark adaptation test protocol can be performed, starting with a luminance below the normal rod threshold and gradually increasing the intensity of the stimulus, ultimately recording a mixed rod / cone response. After a series of dark adaptations, animals can be adapted to a rod-suppressing background light and then a series of light adapting luminances can be performed. If VEPs are to be recorded, given the presence of a functional ERG, subdermal needle or gold cup electrodes can be placed near the inion along the occipital midline (it can be determined which electrode style produces the best recordings in these animals). Placing the recording electrode near the inion has been found to minimize ERG contamination of dog VEPs. If gold cup electrodes are used, the animal's scalp can be shaved at least 1.5 cm on either side of the entire skull midline, wiped with 70% alcohol, and air-dried thoroughly. Conductive electrode paste can be applied to the selected recording site and the cup electrodes can be attached firmly to the skin and held down with medical tape. After the scalp cleaning step, needle electrodes can be inserted subcutaneously without the need for applying electrode paste.

[0233] Electrophysiological data can be quantitatively analyzed. For ERG recordings, the amplitude and latency of the a-wave and b-wave can be recorded and stored in a database. For VEPs, two types of analysis can be used. For flash VEPs, the latency of the N1 and P1 peaks of the response waveform can be measured, as well as the amplitude of these peaks relative to the signal baseline. These parameters can be stored in a database. If pattern reversal VEPs can be recorded, the waveforms can be analyzed using a fast Fourier transform (FFT) referenced to the antiphase frequency of the stimulation pattern to obtain the amplitude and phase components of the steady-state VEP response. These parameters can also be stored in a database to allow easy retrieval of all electrophysiological parameters for each animal as a function of graft type, post-graft duration, and any other relevant treatment parameters. The primary end points of the analysis can be (1) whether visual recovery, defined as light-induced activity in ERG or VEP, occurs after retinal grafting; (2) the type of stem cell treatment (or lack thereof) applied to the animal; and (3) the time until a light-induced response is first observed.

[0234] In vitro multielectrode array (MEA) recordings: In vitro multielectrode array (MEA) recordings can be obtained from the implanted area to directly determine the light response of retinal ganglion cells downstream of the implant. These in vitro recordings require euthanasia of the animal and can be performed at 12 months post-implantation after in vivo functional determinations have been completed. The night before MEA recordings are performed, animals can be dark adapted overnight. The following morning, animals can be euthanized under dim red light, and the eye cups of both eyes are harvested by hemisection of the globe, the anterior half discarded, and the vitreous removed with forceps. The eye cups can be transferred to two capped 50 mL tubes containing Ames medium and continuously supplemented with 95% O25% CO2 using a portable carbogen bath. The capped tubes can be placed in a light-tight box during transport.

[0235] The dog / cat / rabbit eyecups can be transferred to fresh Ames medium and dark adapted for another hour while the transplanted retina is visualized under an infrared viewer to locate the area of ​​transplantation. Once the graft is located, a blade can be used to cut out an approximately 2.5 mm x 2.5 mm eyecup piece containing the transplant. This piece can be placed flat, ganglion cell side down, on a 60-electrode MEA and action potentials from the ganglion cells are recorded extracellularly as previously described. This process does not disturb the attachment of the retina to the pigment epithelium, choroid, and sclera, allowing the transplant to remain firmly attached and the visual cycle responsible for the regeneration of light-activated photopigments is well preserved. 8.6 log to 15.6 log photons cm -2 s -1 A series of intensities of full-field light steps of 1 s duration can be presented. MEA recordings can be taken from retinal areas adjacent to the grafted area as well as from corresponding areas of the non-grafted retina. Both sets of recordings (i.e., graft-containing and control retina) can be spike sorted, for example, using Plexon Offline Sorter software. Alternatively, the amplitude of the light response of each electrode can be easily quantified by calculating the variance of the raw recordings during and 1 s before the onset of the 1 s light stimulation and using the difference between the two variances as the light response amplitude.

[0236] To determine whether the light responses of ganglion cells recorded from the grafted area are significantly greater than those in the control area, the light-induced changes in spike count or recording variance are compared between the two areas, for example by Mann-Whitney U test. For each stimulation intensity, a statistical comparison can be made separately for the following categories of ipRGC light responses: (1) fast excitation at light onset; (2) fast inhibition at light onset; (3) fast excitation at light offset; (4) fast inhibition at light offset; and (5) sluggish excitation, similar to melanopsin-based light responses. If the graft actually enables or enhances the photosensitivity of rod / cone-driven retinal circuits, we might find that the fast light responses are significantly stronger (i.e., categories 1–4) in the grafted area than in the control area. On the other hand, the melanopsin-based light responses may not be significantly affected by the graft, and therefore no differences might be observed.

[0237] For example, if mfERF VEP and pupillometry show improvement in vision in the eye with the graft, behavioral methods for objective vision testing (obstacle courses designed for dogs and cats, and optokinetic tracking for cats) can be implemented.

[0238] Graft-host connectivity can be determined, for example, using: (1) a WGA-HRP transsynaptic tracer expressed by the graft but not by the host cells; (2) IHC / immuno-EM with human (but not cat / dog) cytoplasmic specific antibody SC121 and / or human (but not cat / dog) specific synaptophysin antibody hSYP and / or postsynaptic markers at a site in the recipient retina distant from the human graft, or / and (3) IHC with hSYP+HNu antibodies and retinal cytoplasmic antibodies (e.g., recoverin, CALB2, or / and BRN3A / B) to show human terminal buttons around the (non-human) recipient neurons. Binding can also be demonstrated by injecting the non-viral retrograde tracer cholera toxin B (CtB) into the superior colliculus of recipient animals. The tracer can be injected into the superior colliculus site two weeks before the animals are killed, and IHC can localize CtB in the human graft.

[0239] Multiple pieces of hESC-derived retinal tissue can be placed on a bioprosthetic carrier or scaffold, for example comprising a hydrogel (e.g., HYSTEM®)-based electrospun sheet biomaterial (approximately 3 x 5 mm), or electrospun silk, or other biocompatible material suitable for intraocular implantation as described herein, to create a bioprosthetic retinal patch. The bioprosthetic retinal patch can be implanted subretinally into a subject, who can be followed for a year using the imaging techniques described above, as well as full-field ERG or / and mfERG, and VEP. Behavioral visual testing (obstacle course for dogs and cats, and visual tracking for cats) can also be used.

[0240] A piece of bioprosthetic retina (e.g., 3 x 5 mm) can be implanted into the subretinal space of the model and the grafts can be assessed by in vivo retinal cross-sectional imaging with SD-OCT (and RetCam) at 1 week, 2 weeks, and 1, 2, 4, 6, 9, and 12 months after implantation. Retinal function can be tested in vivo by mfERG (and full-field ERG as well) and vision can be tested by behavioral testing (obstacle course for dogs and cats, and optokinetic tracking for cats), VEP, and pupillometry at 2, 4, 6, 9, and 12 months after implantation. After euthanasia, synapse formation and PR OS outgrowth can be examined by histology and confocal IHC to determine graft integration and connectivity with the host retina. Pre-embedding immuno-EM (synaptic connectivity of the graft to the host) and EM (showing PR outer segments in the graft) can also be used.

[0241] First, bioprosthetic retinas can be implanted into the subretinal space (central retina) of three or more animals. Animals can be immunosuppressed with prednisone + cyclosporine from about -7 days before surgery to about 8 weeks after surgery. Bioprosthetic retinas can be implanted into both eyes of each animal (n=3 grafts, 6 eyes total) by transvitreal subretinal implantation technique. At least one RD animal can be without graft as an untreated control. This method allows for precise delivery of several pieces of hESC-derived retinal tissue into the subretinal space of cats without severe retinal detachment.

[0242] SD-OCT and RetCam imaging can be performed at time=0 (immediately after implantation in the pilot cohort), and at +1 and +2 weeks post-implantation to determine the presence of graft material. This will demonstrate delivery of the sheet-like bioprosthetic grafts to the subretinal space and engraftment of the grafts, resulting in OCT and histology results. Grafts can be monitored for a year or more to generate histological and IHC data on maturation of the hESC-3D retinal tissue within the bioprosthetic retinal patch, as well as on synaptic integration, as well as functional data (mfERG, obstacle course, VEP) on PR function and visual improvement.

[0243] The grafts can be monitored by OCT and the changes in electrical activity by mfERG can be monitored between the grafted site and a site approximately 3-4 mm outside the graft, which can serve as a control set (e.g., a different site in the same retina). At 6-12 months after transplantation, most large eye RD models have a completely degenerated PR layer and a signal detectable by mfERG would be emanating from the graft.

[0244] Table 1: Example of experimental design TIFF2025013961000005.tif102151

[0245] Histology / IHC will provide synaptic connectivity in grafted animals (which can be assessed indirectly during the experiment as a function of mfERG readouts 3-5 months after transplantation, and directly after the animals' death). Transsynaptic tracing and in vivo methods (mfERG, pupil light reflex, visual function tests such as VEP, and visually guided behavioral tests such as mazes can be used. WGA-HRP tracing from human grafts to recipient retinal neurons or / and IHC with SC121, hSYP, HNu, and retinal cell type specific antibodies or / and pre-embedding immuno-EM are all methods to demonstrate functional grafts to host synapses.

[0246] Example 12 Retinal organoids containing hESC-derived retinal tissue (also known as retinal tissue grafts or retinal tissue bioprosthetic grafts or grafts) were implanted into the subretinal space of wild-type cat eyes using a 1.52 mm outer diameter, 1.12 mm inner diameter borosilicate glass cannula (World Precision) after pars plana vitrectomy (n=7 eyes) as described herein at approximately day 40 of differentiation. Group 1 (n=3) received anti-inflammatory doses of prednisone orally throughout the study (5 weeks). Group 2 (n=4) received systemic administration of cyclosporine A in addition to prednisone starting 7 days prior to implantation and continuing throughout the study. Eyes were examined for the presence of subretinal grafts and adverse effects from the surgical procedure by fundus examination and spectral domain optical coherence tomography (OCT).

[0247] The feline retina is structurally similar to the human retina and represents a large-eyed animal model for demonstrating the efficacy of hESC-derived retinal tissue transplantation, as shown in Figure 18. Specifically, cats have a cone-rich region called the central area that resembles the human macula.

[0248] Retinal tissue constructs (organoids) were derived from human embryonic stem cell colonies using different morphogens as described herein. An example of a timeline of retinal differentiation of retinal organoids is shown in Figure 19. As shown in Figures 20A to 20I, immunostaining of retinal organoids with antibodies against retinal progenitor cell markers and early photoreceptor cell markers determined that retinal progenitor cell markers and early photoreceptor markers were expressed in retinal organoids at 8 to 10 weeks.

[0249] Figure 21 shows images of a retinal tissue graft being implanted into the subretinal space of a wild-type cat eye using a glass cannula after pars plana vitrectomy. As shown in Figure 22, a subretinal bleb was formed into which the retinal tissue graft was implanted. Figure 23 shows color fundus and OCT images taken 3 weeks after implantation. These images show the presence and location of the graft in the subretinal space and show that there were no serious adverse effects from the subretinal graft or the surgical procedure.

[0250] Cats were euthanized 5 weeks after graft implantation. Immunohistochemistry (IHC) analysis of retinal sections was performed using human-specific antibodies (e.g., HNu, Ku80, SC121), axon-specific markers, synapse-specific markers, retinal cell type-specific markers, and lymphoid cell and microglia / macrophage markers.

[0251] FIG. 24 shows images of retinal sections from group 1 (+ prednisone, - cyclosporine A) stained with antibodies specific for microglia and macrophages. FIG. 25 shows images of retinal sections taken from group 2 (+ prednisone, + cyclosporine A), also stained with antibodies specific for microglia and macrophages. As shown in FIG. 24 and FIG. 25, the addition of cyclosporine A resulted in reduced accumulation of microglia and macrophages (shown using IBA1 specific staining). In FIG. 25, HNu human specific marker staining of nuclei within the transplanted graft is evident, indicating that the cells of the graft survive at least 5 weeks after transplantation.

[0252] FIG. 26 shows a graph comparing the number of cells positive for microglia and macrophage cell markers in retinal sections of Group 1 (+Prednisone, -Cyclosporin A) and Group 2 (+Prednisone, +Cyclosporin A).

[0253] The location of the graft under the cat retina can also be seen in Figures 27A to 28C. Figure 27A shows a cat retina section of group 2 (+ prednisone, + cyclosporine A) stained with an antibody specific for the photoreceptor marker CRX. Figure 27B shows a cat retina section of group 2 (+ prednisone, + cyclosporine A) stained with a human-specific antibody, HNu. Figure 27C shows a cat retina section of group 2 (+ prednisone, + cyclosporine A) stained with antibodies against both CRX and HNu. As shown, the graft is adjacent to the cat photoreceptor cells. The enlarged inset in Figure 27C shows the cat photoreceptor cells and human cells together. Figure 28A shows a cat retina section of group 2 (+ prednisone, + cyclosporine A) stained with an antibody specific for the retinal ganglion cell (RGC) marker BRN3A. Figure 28B shows a second group of cat retina sections stained for both BRN3A and the human specific marker KU80. Figure 28C shows that the nuclei of the cells are also stained.

[0254] Approximately 5 weeks after transplantation, axonal outgrowth of the transplanted hESC-retinal tissue was shown to be attached to the recipient retina. Figure 29A shows a cat retina section stained with an antibody specific for the calretinin marker CALB2, which is expressed in neurons including the retina. Cells staining positive for CALB2 expression can be seen in Figures 29A, 29B, and 29C. IHC analysis demonstrates that several axons emerging from the transplanted hESC-derived retinal tissue graft are positive for calretinin expression. Figure 29B shows IHC staining for the marker SC121. The antibody against SC121 is specific for the cytoplasm of human cells. Thus, staining with DAPI localizes the axonal outgrowth of the graft relative to the recipient (cat) retinal ganglion cells. IHC analysis shown in FIG. 29C demonstrates that at least 5 weeks after graft implantation, axons from the graft extend and integrate into the external nuclear layer (ONL), the internal nuclear layer (INL), and also into the ganglion cell layer (GCL) of the recipient eye.

[0255] ICH analysis also demonstrated that transplanted human retinal tissue grafts (calretinin positive) that were able to integrate into the retina were also GABAergic, as shown in Figures 30A-C. Figure 30A shows the axons of the retinal graft (stained with an antibody specific for the CALB2 marker) extending toward the cat retina. Figure 30B shows the retinal graft stained with antibodies specific for the human cell markers HNu and CALB2, thereby depicting the graft from the cat retina. The GABA-positive staining of the graft axons shown in Figure 30C further indicates that the axons of the implanted tissue that have integrated into the recipient retina have differentiated to become neurons. These results demonstrate the structural and functional integration of the implanted hESC tissue with the recipient retina.

[0256] ICH analysis also demonstrated that transplanted hESC-derived tissues engrafted without tumors for at least 5 weeks in vivo.

[0257] Example 13 Retinal organoids containing hESC-derived retinal tissue were transplanted into the subretinal or epiretinal space of CRX-mutant cat eyes with retinal degeneration at approximately day 40 of differentiation using a 1.52 mm outer diameter, 1.12 mm inner diameter borosilicate glass cannula (World Precision) after pars plana vitrectomy as described herein. Cyclosporine A was administered systemically throughout the study, starting 7 days prior to transplantation, in addition to an anti-inflammatory dose of orally administered prednisone. OCT images were taken 3 months after implantation of the grafts. Figures 31A to 31G show OCT images of two subjects, demonstrating that hESC-derived retinal tissue grafts transplanted into the subretinal or epiretinal space of a large-eyed animal model (CRX-mutant cat) with retinal degeneration can survive for at least 3 weeks after transplantation.

[0258] Example 14 Referring to FIG. 32, hESC-derived retinal organoids transplanted into the subretinal space of wild-type cats showed BDNF expression 5 weeks after transplantation. As shown, most cells are BDNF+. BDNF is one of the major neurotrophins that support the function of degenerating or damaged neurons. Higher BDNF levels can protect the retina from retinal degeneration due to disease or injury. These results indicate that hESC-derived retinal tissue grafts can provide neurotrophic support to damaged or degenerating retinal tissue after implantation into the intraocular space of a subject's eye.

[0259] From the description herein, it will be understood that the present disclosure encompasses multiple aspects, including, but not limited to, the following.

[0260] 2. A method for one or more of treating retinal damage, slowing the progression of retinal damage, preventing retinal damage, replacing retinal tissue, and repairing damaged retinal tissue, comprising applying hESC-derived retinal tissue to a subject. 3. A method for one or more of slowing the progression of retinal degenerative disease, slowing the progression of retinal degenerative disease after traumatic injury, slowing the progression of age-related macular degeneration (AMD), stabilizing retinal disease, preventing retinal degenerative disease, preventing retinal degenerative disease after traumatic injury, preventing AMD, restoring retinal pigment epithelium (RPE), photoreceptor cells (PRCs), and retinal ganglion cells (RGCs) lost due to disease, injury, or genetic abnormality, increasing RPE, PRCs, and RCGs, or treating RPE, PRC, and RCG deficiency, comprising applying hESC-derived retina to a subject.

[0261] The method of any of the preceding aspects, wherein the retinal damage is due to one or more of blast exposure, genetic disorder, retinal disease, and retinal injury. The method of any of the preceding aspects, wherein the retinal disease comprises a retinal degenerative disease. The method of any of the preceding aspects, wherein the retinal damage is due to one or more of age-related macular degeneration (AMD), retinitis pigmentosa (RP), and Leber's congenital amaurosis (LCA).

[0262] The method of any of the preceding aspects, wherein the hESC-derived retinal tissue comprises retinal pigment epithelial (RPE) cells, retinal ganglion cells (RGC), and photoreceptor (PR) cells. The method of any of the preceding aspects, wherein there is a central core of retinal pigment epithelial (RPE) cells and, radially outward from the RPE cell core, the RPE cells, RGC cells, and PR cells are configured to form a retinal ganglion cell (RGC) layer, a layer of secondary retinal neurons (corresponding to the inner nuclear layer of the mature retina), a photoreceptor (PR) cell layer, and an outer layer of RPE cells. The method of any of the preceding aspects, wherein each of the layers comprises differentiated cells characteristic of cells in a corresponding human retinal tissue layer. The method of any of the preceding aspects, wherein each layer comprises substantially fully differentiated cells.

[0263] The method of any of the preceding aspects, wherein the hESC-derived retinal tissue further comprises a biocompatible scaffold for forming a biological retinal prosthetic device.The method of any of the preceding aspects, wherein the biological retinal prosthetic device comprises between about 10,000 and 100,000 photoreceptors.The method of any of the preceding aspects, wherein the hESC-derived retinal tissue is capable of delivering trophic and neurotrophic factors and mitogens.The method of any of the preceding aspects, wherein the trophic and neurotrophic factors and mitogens comprise one or more of brain-derived neurotrophic factor (BDNF), glial-derived neurotrophic factor (GDNF), neurotrophin-4 (NT4), nerve growth factor-beta (βNGF), and the pro-survival mitogen basic fibroblast growth factor (bFGF=FGF-2).

[0264] The method of any of the preceding aspects, wherein application of the hESC-derived retinal tissue preserves the thickness of the retinal layer for about 1 to about 3 months.The method of any of the preceding aspects, further comprising administration of an immunosuppressant.The method of any of the preceding aspects, wherein the immunosuppressant is administered before, during, and / or after application.

[0265] The method of any of the preceding aspects, wherein the method further comprises modulating intraocular pressure. The method of any of the preceding aspects, wherein the modulating intraocular pressure occurs before, during, and / or after application of the retinal tissue.

[0266] The method of any of the preceding aspects, wherein the tissue is applied using an intraocular transplantation tool.The method of any of the preceding aspects, wherein the hESC-derived retinal tissue is applied subretinal or epiretinal.The method of any of the preceding aspects, wherein applying the hESC-derived retinal tissue results in tumor-free integration of the hESC-derived retinal tissue with the subject's retinal tissue.

[0267] The method of any of the preceding aspects, wherein integration occurs about 4-5 weeks after application. The method of any of the preceding aspects, wherein application does not cause retinal inflammation. The retinal tissue graft of any of the preceding aspects, wherein after application, the retinal tissue undergoes lamination.

[0268] After application, retinal tissue neurons were + and / or K. + The method of any of the preceding aspects, further comprising demonstrating connectivity between the retinal tissue and pre-existing tissue. The method of any of the preceding aspects, wherein the binding is demonstrated by one or more of WGA-HRP transsynaptic tracer, histology, IHC, or electrophysiology. The method of any of the preceding aspects, further comprising measuring a level of functional recovery. The method of any of the preceding aspects, wherein the level of functional recovery comprises an increase in electrophysiological response of at least 75% of baseline.

[0269] A retinal tissue graft for implantation into a subject's eye, comprising retinal pigment epithelial (RPE) cells, retinal ganglion cells (RGCs), secondary retinal neurons, and photoreceptor (PR) cells, the RPE cells, RGC cells, and PR cells configured to form a central core. The retinal tissue graft of any of the preceding aspects, comprising between about 10,000 and 100,000 photoreceptors. The retinal tissue graft of any of the preceding aspects, wherein the secondary retinal neurons correspond to the inner nuclear layer of the mature retina. The retinal tissue graft of any of the preceding aspects, wherein the cells are arranged radially outward from the core, and the retinal tissue comprises a retinal ganglion cell (RGC) layer, a layer of secondary retinal neurons, a layer of photoreceptor (PR) cells, and an outer layer of RPE cells. The retinal tissue graft of any of the preceding aspects, comprising between 5,000 and about 250,000 cells. The retinal tissue graft of any of the preceding aspects, wherein the retinal tissue graft is implanted into the subretinal space or the epiretinal space.

[0270] The retinal tissue graft of any of the preceding aspects, wherein increased synapse formation occurs concomitantly with increased electrical activity.The retinal tissue graft of any of the preceding aspects, wherein after transplantation, neurons connect the graft to existing tissue.The retinal tissue graft of any of the preceding aspects, wherein the neurons are CALB2 positive.The retinal tissue of any of the preceding aspects, wherein connectivity is demonstrated by WGA-HRP transsynaptic tracer.The retinal tissue graft of any of the preceding aspects, wherein after transplantation, axons connect the graft to existing tissue.The retinal tissue of any of the preceding aspects, wherein the axons are CALB2 positive.The retinal tissue graft of any of the preceding aspects, wherein after transplantation, cells of the graft mature into RGCs.

[0271] The retinal tissue graft of any of the preceding aspects, which forms synapses with existing neurons after transplantation.The retinal tissue graft of any of the preceding aspects, which forms a bond with existing tissue after transplantation.The retinal tissue of any of the preceding aspects, which forms a bond 1 day to about 5 weeks after transplantation.The retinal tissue graft of any of the preceding aspects, which forms an axon that spans the existing tissue ONL after transplantation.

[0272] The retinal tissue graft of any of the preceding aspects, which produces a paracrine factor.The retinal tissue graft of any of the preceding aspects, wherein the paracrine factor is produced before and / or after application.The retinal tissue graft of any of the preceding aspects, which produces a neurotrophic factor.The retinal tissue graft of any of the preceding aspects, which produces a neurotrophic factor before or after application.The retinal tissue of any of the preceding aspects, wherein the neurotrophic factor comprises one or more of BDNS, GDNF, bNGF, NT4, or bFGF.

[0273] The retinal tissue graft of any of the preceding aspects, wherein after transplantation, the level of functional recovery is measured as an increase in electrophysiological response.The retinal tissue graft of any of the preceding aspects, wherein the level of functional recovery is measured as an increase in electrophysiological response to at least 10% of baseline.The retinal tissue graft of any of the preceding aspects, wherein after transplantation, the axons of the graft are integrated into existing tissue.

[0274] In the claims, reference to an element in the singular is intended to mean "one or more" and not "one and only one" unless otherwise specified. All structural, chemical, and functional equivalents of the elements of the embodiments of the present disclosure known to those of skill in the art are expressly incorporated herein by reference and are intended to be encompassed by the claims. Furthermore, no element, component, or method step of the present disclosure is intended to be dedicated to the public, even if such element, component, or method step is expressly recited in the claims. No element of the claims herein is construed as a "means-plus-function" element unless expressly recited by the phrase "means for". No element of the claims herein is construed as a "step-plus-function" element unless expressly recited by the phrase "step for".

Claims

1. A method for one or more of treating retinal damage, slowing the progression of retinal damage, preventing retinal damage, replacing retinal tissue, and repairing damaged retinal tissue, comprising applying an hESC-derived retinal tissue graft to a subject.

2. 1. A method for one or more of slowing the progression of a retinal degenerative disease, slowing the progression of a retinal degenerative disease after traumatic injury, slowing the progression of age-related macular degeneration (AMD), slowing the progression of an inherited retinal disease, stabilizing a retinal disease, preventing a retinal degenerative disease, preventing a retinal degenerative disease after traumatic injury, improving vision or visual perception, preventing AMD, restoring retinal pigment epithelium (RPE), photoreceptor cells (PRCs), and retinal ganglion cells (RGCs) lost due to disease, injury, or genetic abnormality, increasing RPE, PRCs, and RCGs, or treating RPE, PRC, and RCG deficiencies, comprising applying an hESC-derived retinal tissue graft to a subject.

3. 10. The method of claim 1, wherein the retinal damage is caused by one or more of blast exposure, a genetic disorder, a retinal disease, and a retinal injury.

4. 4. The method of claim 3, wherein the retinal disease comprises a retinal degenerative disease.

5. 2. The method of claim 1, wherein the retinal damage is caused by one or more of age-related macular degeneration (AMD), retinitis pigmentosa (RP), and Leber congenital amaurosis (LCA).

6. 3. The method of claim 1 or 2, wherein the hESC-derived retinal tissue comprises retinal pigment epithelial (RPE) cells, retinal ganglion cells (RGCs), and photoreceptor (PR) cells.

7. The method of claim 6, wherein the RPE cells, the RGC cells, and the PR cells are configured such that there is a central layer of RPE cells and radially outward from the RPE cell layer there is a retinal ganglion cell (RGC) layer, a layer of secondary retinal neurons (corresponding to the inner nuclear layer of the mature retina), a photoreceptor (PR) cell layer, and an outer layer of RPE cells.

8. The method of claim 7, wherein each of said layers comprises differentiated cells characteristic of cells in a corresponding human retinal tissue layer.

9. The method of claim 7, wherein each of said layers comprises progenitor cells, and after application, some or all of said progenitor cells differentiate into mature cells of the corresponding human retinal tissue layer.

10. The method of claim 7, wherein the layer comprises substantially fully differentiated cells.

11. The method of claim 1 or 2, wherein the hESC-derived retinal tissue further comprises a biocompatible scaffold for forming a bioprosthetic retinal patch.

12. The method of claim 7, wherein the bioprosthetic retinal graft comprises between about 10,000 and 100,000 photoreceptor cells.

13. The method of claim 11, wherein several of the hESC-derived retinal tissue fragments are added to the biocompatible scaffold to form a large bioprosthetic patch.

14. The method of claim 6, wherein the hESC-derived retinal tissue graft or dissociated cells of the hESC-derived retinal tissue graft are capable of delivering one or more of neurotrophic factors, neurotrophic exosomes, and mitogens to a subject.

15. 15. The method of claim 14, wherein the neurotrophic factors and mitogens comprise one or more of brain-derived neurotrophic factor (BDNF), glial-derived neurotrophic factor (GDNF), neurotrophin-34 (NT34), neurotrophin 4 / 5, nerve growth factor-beta (βNGF), proNGF, PEDF, CNTF, the pro-survival mitogen basic fibroblast growth factor (bFGF=FGF-2), and a pro-survival member of the WNT family.

16. 3. The method of claim 1 or 2, wherein application of the hESC-derived retinal tissue graft preserves the thickness of the retinal layer at the site of application for about 1 to about 3 months.

17. 3. The method of claim 1 or 2, further comprising administration of an immunosuppressant.

18. The method of claim 1 or 2, further comprising administering epinephrine before, during and / or after application of the retinal graft.

19. 20. The method of claim 17, wherein the immunosuppressant is administered before, during, and / or after the administering.

20. The method of claim 1 further comprising modulating intraocular pressure.

21. 21. The method of claim 20, wherein the adjustment of intraocular pressure occurs before, during, and / or after application of the retinal tissue.

22. The method of claim 1 , wherein the tissue is applied using an intraocular implantation tool.

23. The method of claim 1 or 2, wherein the hESC-derived retinal tissue is applied subretinal or epiretinal.

24. 3. The method of claim 1 or 2, wherein application of the hESC-derived retinal tissue graft results in tumor-free integration of the hESC-derived retinal tissue with the subject's retinal tissue.

25. 25. The method of claim 24, wherein integration of the retinal graft occurs about 2 to 10 weeks after application.

26. 26. The method of claim 25, wherein the integration comprises structural integration.

27. 25. The method of claim 24, wherein said integration comprises structural integration and occurs about 1 to 6 months after application.

28. 10. The method of claim 1, wherein application does not cause retinal inflammation.

29. 27. The retinal tissue graft of claim 26, wherein after application, the retinal tissue undergoes lamination.

30. After application, the retinal tissue neurons + , K + and / or Ca ++ The method of claim 1 , further comprising providing an indication of a current.

31. The method of claim 1, further comprising demonstrating connectivity between the retinal tissue and pre-existing tissue.

32. 32. The method of claim 31, wherein said binding is demonstrated by one or more of WGA-HRP transsynaptic tracer, histology, IHC, or electrophysiology.

33. 10. The method of claim 1, further comprising measuring a level of functional recovery.

34. 34. The method of claim 33, wherein the level of functional recovery comprises an increase in electrophysiological response of at least 10% above baseline.

35. 1. A retinal tissue graft for transplantation into an eye of a subject, comprising: A retinal tissue graft comprising retinal pigment epithelial (RPE) cells, retinal ganglion cells (RGCs), second order retinal neurons, and photoreceptor (PR) cells, wherein the RPE cells, the RGC cells, and the PR cells are configured to form a central core.

36. The retinal tissue graft of claim 35, having between about 1,000 and 250,000 photoreceptors.

37. The retinal tissue graft of claim 35, wherein said second order retinal neurons correspond to the inner nuclear layer of a mature retina.

38. The retinal tissue graft of claim 35, wherein the cells are arranged radially outward from the core, and the retinal tissue comprises a retinal ganglion cell (RGC) layer, a layer of secondary retinal neurons, a layer of photoreceptor (PR) cells, and an outer layer of RPE cells.

39. 36. The retinal tissue graft of claim 35, comprising between 1,000 and about 250,000 cells.

40. The retinal tissue graft of claim 35, which is implanted into the subretinal space or epiretinal space.

41. The retinal tissue graft of claim 40, wherein said retinal tissue graft is implanted into the subretinal or epiretinal space near the macula.

42. The retinal tissue graft of claim 35, wherein the increased synapse formation occurs concomitantly with increased electrical activity.

43. The retinal tissue graft of claim 35, wherein after transplantation, neurons connect the graft to existing tissue.

44. The retinal tissue graft of claim 43, wherein said neurons are CALB2 positive.

45. 44. The retinal tissue of claim 43, wherein the binding is demonstrated by a WGA-HRP transsynaptic tracer.

46. The retinal tissue graft of claim 35, wherein after implantation, axons connect the graft to existing tissue.

47. The retinal tissue of claim 46, wherein the axons are CALB2 positive.

48. The retinal tissue graft of claim 35, wherein after transplantation, the cells of the graft mature into RGCs.

49. The retinal tissue graft of claim 35, which forms synapses with existing neurons after transplantation.

50. The retinal tissue graft of claim 35, which forms a bond with existing tissue after transplantation.

51. The retinal tissue of claim 50, wherein the bond forms between 1 day and about 5 weeks after transplantation.

52. The retinal tissue graft of claim 35, which, after transplantation, forms axons that span the existing ONL tissue.

53. The retinal tissue graft of claim 35, which produces a paracrine factor.

54. The retinal tissue graft of claim 53, wherein the paracrine factors are produced before and / or after application.

55. The retinal tissue graft of claim 35, which produces a neurotrophic factor.

56. The retinal tissue graft of claim 55, which produces a neurotrophic factor before or after application.

57. 56. The retinal tissue of claim 55, wherein the neurotrophic factors comprise one or more of BDNS, GDNF, bNGF, NT4, bFGF, NT34, NT4 / 5, CNTF, PEDF, serpins, or WNT family members.

58. The retinal tissue graft of claim 35, wherein after transplantation, the level of functional recovery is measured as an increase in electrophysiological response.

59. The retinal tissue graft of claim 58, wherein the level of functional recovery is measured as an increase in electrophysiological response to at least 10% of baseline.

60. 36. The retinal tissue graft of claim 35, wherein after implantation, the axons of the graft penetrate and integrate into existing tissue.

61. The retinal tissue graft of claim 35, wherein said tissue is derived from human pluripotent stem cells.

62. The retinal tissue graft of claim 35, which is useful for slowing the progression of a retinal degenerative disease, slowing the progression of a retinal degenerative disease after traumatic injury, slowing the progression of age-related macular degeneration (AMD), slowing the progression of an inherited retinal disease, stabilizing a retinal disease, preventing a retinal degenerative disease, preventing a retinal degenerative disease after traumatic injury, improving vision or visual perception, preventing AMD, restoring retinal pigment epithelium (RPE), photoreceptor cells (PRCs), and retinal ganglion cells (RGCs) lost due to disease, injury, or genetic abnormality, increasing RPE, PRCs, and RCGs, or treating RPE, PRC, and RCG deficiency in a subject.

63. The retinal tissue graft of claim 35, wherein after implantation into the intraocular space of a recipient, the PR and RPE layers are layered and developed, including PR outer segment elongation, synapse formation, electrophysiological activity, and connectivity with recipient retinal cells, and is capable of tumor-free engraftment for at least about 6 to 24 months.

64. The retinal tissue graft of claim 35, which is capable of extending axons into the outer nuclear layer (ONL), inner nuclear layer (INL), and ganglion cell layer (GCL) of the recipient and capable of integrating the axons into said layers 5 weeks after implantation into the intraocular space of the recipient eye.

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