WO2003018040A1

WO2003018040A1 - Composite graft for treatment of retinal diseases

Info

Publication number: WO2003018040A1
Application number: PCT/US2002/026859
Authority: WO
Inventors: Michael J. Young; Henry J. Klassen
Original assignee: The Schepens Eye Research Institute, Inc.
Priority date: 2001-08-24
Filing date: 2002-08-23
Publication date: 2003-03-06
Also published as: WO2003018040A9

Abstract

The invention is directed to a composite graft for the treatment of conditions associated with photoreceptor loss. Such conditions include, for example, central vision loss as a result of age-related macular degeneration (AMD). In particular, the composite graft of the invention comprises a layer of connecting cells and a layer of photoreceptors. The connecting cell layer synapse locally with the adjacent photoreceptors of the graft and, after transplantation, with interneurons or retinal ganglion cells of the recipient retina, functionally linking the grafted photoreceptors with the host's visual system.

Description

TITLE OF THE INVENTION COMPOSITE GRAFT FOR TREATMENT OF RETINAL DISEASES

CROSS REFERENCE TO RELATED APPLICATIONS This application claims the benefit under 35 U.S.C. §119 (e) of U.S. Provisional Patent Application No. 60/314,911, filed August 24, 2001, entitled COMPOSITE GRAFT FOR TREATMENT OF RETINAL DISEASES, and U.S. Provisional Patent Application No. 60/343,402, filed December 21, 2001, also entitled COMPOSITE GRAFT FOR TREATMENT OF RETINAL DISEASES, the whole of which are hereby incorporated by reference herein.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR

DEVELOPMENT N/A

BACKGROUND OF THE INVENTION Many parts of the central nervous system (CNS) exhibit laminar organization, and neuropathological processes generally involve more than one of these multiple cellular layers. Diseases of the CNS frequently include neuronal cell loss, and, because of the absence of endogenous repopulation, effective recovery of function following CNS-related disease is either extremely limited or completely absent. In particular, the common retinal condition known as age-related macular degeneration (AMD) results from the loss of photoreceptors together with the retinal pigment epithelium (RPE) , with additional variable involvement of internuncial ("relay") neurons of the inner nuclear layer (INL). Restoration of moderate-to-high acuity vision, therefore, requires the functional replacement of all the damaged cellular layers.

AMD is a clinical diagnosis encompassing a range of degenerative conditions that likely differ in etiology at the molecular level. All cases of AMD share the feature of photoreceptor cell loss within the central retina. However, this common endpoint appears to be a secondary consequence of earlier abnormalities at the level of the RPE and underlying Bruch' s membrane. The latter may relate to difficulties with photoreceptor membrane turnover, which are as yet poorly understood.

An additional feature present in some cases of AMD is the presence of aberrant blood vessels, which result in a condition known as choroidal neovascularization (CNV) . This neovascular ("wet") form of AMD is particularly destructive and seems to result from a loss of integrity of Bruch' s membrane. Breaks in Bruch' s membrane allow new vessels from the choroidal circulation access to the subretinal space, where they physically disrupt outer-segment organization and cause vascular leakage or hemorrhage leading to additional photoreceptor loss (see Fig. 3, showing fibrovascular fronds 18 of new choroidal vessels in the macula) .

CNV is readily destroyed by laser treatment. Thus, laser treatment for the "wet" form of AMD is in general use in the United States. There are often undesirable side effects, and therefore, patient dissatisfaction, however, with treatment outcome. This is due to the fact that laser burns, if they occur, are associated with photoreceptor death and, therefore, with absolute, irreparable blindness within the corresponding part of the visual field. In addition, laser treatment does not fix the underlying predisposition towards developing CNV. Indeed, laser burns have been used as a convenient method for induction of CNV in monkeys (Archer and Gardiner, 1981) . Macular laser treatments for CNV are used much more sparingly in other countries such as the U.K. There is no generally recognized treatment for the more common "dry" form of AMD, in which there is photoreceptor loss overlying irregular patches 14 of RPE atrophy 16 in the macula (see Fig. 2) .

In addition to AMD, a variety of other degenerative conditions affecting the macula include, but are not limited to, cone dystrophy, cone-rod dystrophy, malattia leventinese, Doyne honeycomb dystrophy, Sorsby' s dystrophy, Stargardt disease, pattern/butterfly dystrophies, Best vitelliform dystrophy, North Carolina dystrophy, central areolar choroidal dystrophy, angioid streaks, and toxic maculopathies. General retinal diseases that can secondarily effect the macula include retinal detachment, pathologic myopia, retinitis pigmentosa, diabetic retinopathy, CMV retinitis, occlusive retinal vascular disease, retinopathy of prematurity (ROP) , choroidal rupture, ocular histoplasmosis syndrome (POHS) , toxoplasmosis, and Leber's congenital amaurosis. None of the above lists is exhaustive.

All of the above conditions involve loss of photoreceptors and, therefore, treatment options are few and insufficient. Although new photoreceptors (PRCs) have been introduced experimentally by transplantation, grafted PRCs show a marked reluctance to link up with surviving neurons of the host retina. In addition, delivery of RPE cells or iris epithelial cells as suspensions or loose sheets to the subretinal (submacular) space of the diseased human eye has been described in Algvere et al. (1994), Algvere et al. (1997), Algvere et al. (1999), eisz et al. (1999), Thumann et al . (2000), and del Priori et al. (2001). Pigment epithelial cells (RPE or IPE) delivered as a suspension do not distribute evenly, do not form an intact monolayer and have not been shown to be beneficial in the treatment of human disease. On the contrary, ectopic RPE cells can behave like fibroblasts and have been associated with a number of destructive retinal complications including axonal loss (Villegas-Perez, et al, 1998) and proliferative vitreoretinopathy (PVR) with retinal detachment (Cleary and Ryan, 1979) . RPE delivered as a loose sheet tends to scroll up. This results in poor effective coverage of photoreceptors as well as a multilayered RPE with incorrect polarity, possibly resulting in cyst formation or macular edema. Thus, neither approach in which RPE is used alone effectively reconstitutes an intact pigmented epithelium or replaces photoreceptors .

Delivery of neural retinal grafts to the subretinal (submacular) space of the diseased human eye has been described in Kaplan et al. (1997), Humayun et al. (2000), and del Cerro et al. (2000). Neural retinal grafts delivered as micro-aggregates of neural retinal cells generally form rosettes, not a layered retina. A more serious problem is that neural retinal grafts typically do not functionally integrate with the host retina. In addition, the absence of an intact RPE monolayer means that RPE dysfunction or disruption of Bruch' s membrane has not been rectified. Both are fundamental antecedents of visual loss. Neural retinal grafts delivered as full thickness retinal grafts are laminar but have too many layers. The supernumerary layers pose a formidable barrier to meaningful synaptic integration between photoreceptors of the graft and interneurons of the host .

Co-transplantation of RPE and neural retina to the subretinal space in animal models is described in Seiler et al. (1995), Aramant et al. (1999), and Sharma et al. (2000). Co- transplants including both neural and RPE elements often exhibit the same problems described in detail above. Furthermore, despite the presence of both neural retina and RPE there remains the essential problem of a functional disconnection between the graft and the host retina. BRIEF SUMMARY OF THE INVENTION The invention is related to a method and a composite graft for functionally restoring vision to a patient with a condition associated with central vision loss as a result of age-related macular degeneration, other degenerations of the macula, or other diseases involving loss of photoreceptor cells of the retina. In particular, the composite graft of the invention comprises a layer of connecting cells that are able to form functional connections between a separate layer of photoreceptors in the graft and interneurons of the host retina. Exemplary connecting cells include, but are not limited to, neural stem cells, such as retinal stem cells or brain stem cells, or more differentiated neural progenitor types such as to give rise to bipolar cells in the retina. The connecting cells synapse locally with the adjacent photoreceptors of the graft and, after transplantation, with interneurons or retinal ganglion cells of the recipient retina, thereby functionally linking the grafted photoreceptors with the host's visual system. In one aspect, a layer equivalent to or precursor to a retinal pigment epithelial (RPE) monolayer is also included in the graft. In another aspect of the composite graft of the invention, the layer of RPE cells includes a membrane, such as Bruch' s membrane, or an artificial equivalent of Bruch' s membrane, to provide a growth substrate for the RPE cells, for support of the graft during preparation, for aiding delivery to the transplantation site, to restore structural integrity to the host RPE layer and to provide a barrier to destructive neovascular elements from the host choroidal circulation. In yet another aspect of the invention, the support membrane is in the form of a scaffold or a biodegradable polymer, which also may be used for the delivery of a variety of compounds including angiogenic or antiangiogenic agents. BRIEF DESCRIPTION OF THE FIGURES Other features and advantages of the invention will be apparent from the following description of the preferred embodiments thereof and from the claims, taken in conjunction with the accompanying drawings, in which:

FIG. 1A illustrates a cut-away view of a normal fundus in relation to the eye;

FIG. IB illustrates a partial view of the normal fundus showing the vessels of the retinal vascular arcades radiating from the optic disc. The temporal arcades encompass the clinical macula (dotted circle) and fovea (central dimple) ;

FIG. 2 illustrates a "Dry" age-related macular degeneration (AMD) in the fundus, showing irregular ("geographic") patches of retinal pigment epithelium atrophy (stippled) involving the macula;

FIG. 3 illustrates a "Wet" AMD where fibrovascular fronds of new choroidal vessels are present under the macula;

FIG. 4 illustrates a delivery of a foldable version of a composite graft to the subretinal space using a soft-tipped cannula via a pars plana approach;

FIG. 5 illustrates a composite graft centered under the macula in the subretinal space. The closed retinotomy is visible temporally; FIG. 6 illustrates a cross-sectional view of a composite graft transplanted under the diseased macula. The graft is outlined in heavy black box, and has yet to integrate with the host tissues. The choriocapillaris is absent in the diseased area and the neural stem cells are present in the top (inner, vitread) layer of the graft; FIG. 7A shows a thin section of fixed human retinal tissue from the parafoveal region 7, modified from Dowling (1987) . This is the outer retina layer from a donor retinal tissue;

FIG. 7B shows a thin section of fixed human retinal tissue from the parafoveal region 7, modified from Dowling (1987) . This is the outer plexiform layer from a donor retinal tissue;

FIG. 7C shows a thin section of fixed human retinal tissue from the parafoveal region 7, modified from Dowling (1987) . This is the retinal degeneration in the inner nuclear and plexiform layers and the ganglion cell layer in AMD;

FIG. 8 shows an in vivo composite graft of neural stem cells and the integration of photoreceptors; and

FIG. 9 shows a phase-contrast photomicrograph of an example of retinal pigment epithelium-retinal stem cell composite in culture (magnification 10X) ;

FIG. 10 shows a fluorescent photomicrograph of the generation of bi-polar cells from stem cells in vi tro (20X) ;

FIG. 11 shows a fluorescent photomicrograph of the result of grafting the cells obtained from FIG. 10 into a retinal explant (2OX) ;

FIG. 12 shows a photograph of an exemplary standard pars plana vitrectomy with a composite graft consisting of a biodegradable polymer and murine retina stem cells;

FIG. 13 shows a fluorescent photomicrograph of the composite graft from FIG. 12 five weeks after transplantation (20X) ; and

FIG. 14 shows a fluorescent photomicrograph of a composite graft without a biodegradable polymer five weeks after grafting (20X) .

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to treating a patient with conditions associated with photoreceptor loss using the composite graft and method of the invention. Such conditions associated with photoreceptor loss, include, but are not limited to, retinal degeneration and retinal detachment. This invention provides a method of functionally connecting grafted photoreceptors (PRCs) with the host visual pathway to effect long-term PRC replacement and, thus, establish, improve or restore vision. In addition, the long-term function of PRCs requires a healthy blood supply and retinal pigment epithelial (RPE) cells as well as an intact Bruch' s membrane to eliminate aberrant neovascular growth. Because these structures are compromised or disrupted in some degenerative conditions such as AMD, the method of the invention also entails restoration of the underlying RPE, Bruch' s membrane and choriocapillaris when required for therapeutic efficacy.

The composite graft of the invention comprises a layer of photoreceptors and a layer of connecting cells. The connecting cells are able to send neural processes into, and form connections with, the photoreceptor layer of the graft and the surviving interneurons or retinal ganglion cells of the recipient retina. Exemplary connecting cells include, but are not limited to, a range of phenotypically plastic cells of neural lineage as typified by neural stem cells derived from, e.g., either the brain or retina. Also included are more differentiated, yet not fully mature, neural progenitor cells such as those predisposed to differentiate into retinal bipolar cells as well as less differentiated cells such as embryonic stem cells which can be directed towards a neural and/or retinal fate. Additional cells of interest include stem cells of various types such can be derived from bone marrow or from fat, skin, other tissues or tumors, or that might be generated through pharmacological treatment, genetic manipulation, or nuclear transfer. For example, neural stem cells synapse locally with the adjacent photoreceptors of the graft and, after transplantation, with interneurons of the recipient retina, thereby functionally linking the grafted photoreceptors with the host's visual system. In another form of the invention, connecting cells would not be directly supplied as a cellular layer but would be induced by bioactive agents contained within the graft. These agents would act on surviving host neurons, particularly bipolar cells, to induce neurite sprouting and synapse formation with the photoreceptors of the graft. One such possible agent is epidermal growth factor (EGF) .

In one aspect, a layer equivalent to or precursor to a RPE monolayer is also included in the graft. In another aspect, a layer of RPE cells along with a supporting membrane such as Bruch' s membrane is included in the graft. In yet another aspect of the invention, the supporting membrane, used to provide a growth substrate for the RPE cells, for support of the graft during preparation, for aiding delivery to the transplantation site, to restore structural integrity to the host RPE layer and to provide a barrier to destructive neovascular elements from the host choroidal circulation, is in the form of a stable scaffold or a biodegradable polymer, which also may be used for delivery of bioactive compounds such as angiogenic or antiangiogenic agents along with the graft.

The components of the composite graft of the invention can be obtained most easily from a variety of mammalian donor eyes. Sources of the different layers may be derived from healthy mammals, embryos, cadavers, or surgical specimens. An efficient method of obtaining the composite graft of the invention is to extract three layers of the graft from donor eyes - the RPE layer, Bruch' s membrane and the photoreceptor layer - and then to lay down the connecting cell layer in cell culture. Fig. 1 depicts the region of the eye undergoing treatment. As shown in Fig. 1A, the fundus region 4 is depicted in relation to the eye 2. In Fig. IB, the vessels 12 of the retinal vascular arcades radiate from the optic disc 10. The temporal arcades encompass the clinical macula 6 and the fovea 8, which is the area of highest visual acuity.

Currently, there is no recognized restorative treatment for photoreceptor loss, either in the macula or elsewhere in the retina. Experimental therapies have failed to deliver reproducible clinical improvements, as described above. The intimate relationship between PRCs and RPE is such that loss of one of these layers due to a pathological process frequently results in focal disruption or loss of the other. Because central visual deficits typically involve both layers, and because the survival and laminar organization of the two layers are somewhat interdependent, the present invention has identified the need for simultaneous replacement of both PRCs and RPE when reconstructing the central retina, particularly in the setting of macular degeneration. Moreover, the present invention reflects the fact that high acuity vision (>20/200) requires precise cytoarchitectural organization of both the RPE and PRC layers in the foveal region. In particular, RPE cells must take the form of an epithelial monolayer adhering to an intact basement membrane. The RPE cells must be joined by tight junctions and show the appropriate polarity, i.e., apical surfaces facing the photoreceptors and basal surfaces facing Bruch' s membrane. High acuity vision requires that the photoreceptors be numerous and that they be cones. Outer segments must be properly aligned and closely packed in a regular mosaic. Note that a modified version of the invention without an RPE component is indicated when the host RPE layer remains intact, as is present in many cases of retinal detachment and retinitis pigmentosa. Additionally, the present invention solves the problem of re-establishing visual benefit after treatment for age-related macular degeneration (AMD) . Current animal data in the field of retinal transplantation have revealed that grafted PRCs will survive. However, they fail to establish synaptic connections with host retinal cells, conferring no reproducible visual benefit upon the recipient. In contrast, the present study with neural stem cell (NSC) transplantation indicates that NSCs show a high capacity for integration with the mature mammalian retina, capable of providing the required functional linkage between grafted photoreceptors and the damaged retina, even in mature recipients. The strategy contemplated by the method of the invention is to create ex vivo a composite cellular graft comprising at least a connecting cell layer and a photoreceptors layer. In an exemplary cellular graft, as shown in Fig. 6, a composite graft 24 comprises an RPE monolayer 32 and Bruch' s membrane 33, a dense layer of photoreceptors 28, which includes the outer nuclear layer 50 and cones 30, and a layer of neural stem cells 26 located at the level of the outer plexiform layer 48 (see Fig. 6, as shown in the heavy black box) . The graft is placed in the macula, positioned in the subretinal space between the remaining neural retina and choroid. The remaining layers of the host retina are, starting from the vitreal surface 37, the nerve fiber layer 40, the ganglion cell layer 42, the inner plexiform layer 44, and the inner nuclear layer 46. Within the graft, the neural stem cells 26 are located in the innermost (top) layer, corresponding in position to the host outer plexiform layer 48. In addition, the graft is designed to induce a new choriocapillaris (CC) from the remaining choroidal vasculature 36 of the host, which will be maintained by the grafted RPE layer 32. Together, these structures (RPE and CC) will maintain a functional Bruch' s membrane, thereby preventing ingress of new vessels to the subretinal space. The RPE also maintains photoreceptor viability and function. The neural stem cells synapse locally with grafted photoreceptors 28 on the one hand, and interneurons of the host on the other, providing the essential functional neuronal linkage between graft and host.

In the preferred embodiment, the graft of the invention { see Fig. 6) uses a common basement membrane, e.g., Bruch' s membrane 33, as a physical barrier against neovascular ingrowth and also to serve as an adherent substrate for the RPE layer. Adjacent to the RPE lies a highly anastomotic network of capillaries known as the choriocapillaris (CC) 34. The choroidal vasculature and CC supply the metabolic needs of the PRCs, as well as the outer portion of the inner nuclear layer. The reconstructed RPE and CC provide the maintenance required for the restored Bruch 's membrane. Exemplary membranes include, but are not limited to, donated Bruch' s membrane, either allogeneic, xenogeneic or possibly autologous; alternative biologic membranes, such as Descemet's, amniotic or lens capsule (Nicolini et al, 2000; Koizumi et al . , 2000; Thumann, et al, 1997); an artificial substrate manufactured to provide a suitable temporary scaffold or a biodegradable polymer until the combined actions of the reconstructed RPE and CC are sufficient to generate a new Bruch' s membrane de novo. Such a substrate can also serve as a particularly convenient delivery system for angiogenic/anti-angiogenic agents, as discussed above, or any additional desirable agents. In the most simple example, a graft consisting of a photoreceptor sheet embedded in a biodegradable matrix containing one or more bioactive agents would be delivered to the subretinal space of a recipient with PRC loss but intact RPE layer. The bioactive agents would serve as a virtual connecting cell layer by inducing neurite outgrowth, synaptogenesis, and possibly transient dedifferentiation of the host bipolar cells, resulting in connectivity between graft and host. The list of agents of potential interest in this regard includes, but is not limited to, epidermal growth factor (EGF) , transforming growth factor (TGF) -alpha, ciliary neurotrophic factor (CNTF) , neurotrophin-3 (NT-3) , nerve growth factor (NGF) , brain-derived neurotrophic factor (BDNF) , glial-derived neurotrophic factor (GDNF) , beta fibroblast growth factor (bFGF) , sonic hedgehog, Interleukin (IL)-l-beta, IL-6, and TGF-beta.

The CC is frequently disrupted or even eliminated in AMD, particularly late in the course of the disease or following macular laser treatments. The RPE is known to play an inductive role in the formation of the CC, and this effect has been attributed to secretion of the growth factor VEGF (Blaauwgeers et al, 1999) . The present invention is designed to ensure an adequate blood supply to the reconstructed macula, indirectly provided by the restored RPE of the invention.

An additional feature that can be incorporated into the graft, as needed, is the use of recombinant growth factors, particularly vascular endothelial growth factor (VEGF) , but also including basic fibroblast growth factor (bFGF) or other angiogenic agents. These molecules can be positioned on the side of the graft facing the sclera (i.e., sclerad) , preferably attached or embedded in such a way as to not be transferred to the inward-facing (vitread) surface while the graft is scrolled up within the cannula during delivery (Fig. 4). Positioning molecules of interest on one side of the graft allows these molecules to exert an influence on one side of the graft alone. This can be of crucial importance, particularly with respect to angiogenesis, which is needed below Bruch' s membrane and highly undesireable above it. Furthermore, the growth factor can be present in a gradient that is greatest centrally on one of the graft's surfaces and decreases towards the perimeter. Anti-angiogenic agents such as anti-VEGF antibodies can be positioned within the graft so as to discourage new vessels from growing around the perimeter into the subretinal space, or through the new Bruch' s membrane and generating an RPE detachment.

The RPE cells of the invention are delivered as an intact epithelium. The cells form tight junctions with each other and exhibit a clearly defined polarity with distinct apical and basal surfaces (either before or shortly after transplantation) . Polarity is frequently lost when RPE cells are cultured as a monolayer, yet is essential for some important RPE cell functions, particularly the active movement of fluid from vitreous to choroid that maintains retinal attachment, but also for the catabolism of photoreceptor outer segment membranes. Tight junctions are also important to the blood-retinal barrier. The general integrity of the RPE as an epithelial monolayer is important for keeping RPE cells from migrating in destructive ways and resisting neovascular incursion. In the preferred method of the invention, the RPE is replaced as an epithelial monolayer with the correct polarity. RPE can be harvested as a sheet from donor eyes, either, allogeneic, xenogeneic or autologous . One method includes harvesting RPE, Bruch' s membrane, and photoreceptors simultaneously from a donor eye without disturbing the layers in their native apposition. Alternatively, RPE cells can be proliferated in culture and secondarily grown as a monolayer. RPE cells can be generated from other cells, such as stem cells (e.g., retinal, neural, or embryonic), and grown as a monolayer, either before (Fig. 9) or after (Fig. 14) transplantation.

Several parameters are required for acuity and visual image resolution. In particular, the density and alignment of the photoreceptors present in the central retina must be optimized when reconstructing the macula. Although the retina contains both types of photoreceptors, namely rods and cones, it is cones that are of particular importance to high acuity vision. In fact, the central macula is devoid of rods, and the normal pattern of distribution will inform the construction of the photoreceptor layer graft. In the invention, the PRCs are replaced as an organized sheet, sandwiched between the RPE and stem cell layers of the composite graft. Sources for PRCs include, but are not limited to, donated human eyes and non- human primates. In addition, non-primate species, such as a pig, could be genetically modified to "humanize" the retina or to develop a focal density of cones that can be harvested for transplantation. Another parameter of interest is the age of the donor tissue. The most facile approach is to harvest the central retina at a time after the photoreceptor layer has developed. In this way, the desired cell type is assured and the appropriate outer segment alignment has already been generated by innate mechanisms. Simultaneously harvesting PRCs and RPE as intact, mutually adherent sheets preserves this delicate alignment. It is also desirable to harvest PRCs at an early age, (e.g., prenatal) in order to maximize their innate developmental plasticity in terms of synapse formation. Alternatively, tissue can be harvested earlier or later, in which case it may be desirable to supplement the graft with additional factors or agents, for example, to induce the desired phenotype in developing PRCs. Another potential source of PRCs is from retinal stem cells (RSCs) . RSCs can be grown in culture and differentiated into photoreceptors. In particular, RSCs can be grown within an artificial substrate at high density and induced to differentiate along the cone pathway, e.g., using sonic hedgehog (SHH) . The substrate can- be seeded while already in contact with an RPE layer or juxtaposed secondarily. Cones might also be obtainable from other types of cells, particularly neuronal stem cells, embryonic stem (ES) cells, or transdifferentiated cells. When using cultured cells, such as RSCs, it may again be desirable to supplement the graft with factors or agents that promote the induction of the desired phenotype or inhibit the development of undesirable phenotypes. Other supplemental factors of great interest are those that enhance cell survival. This applies to all layers of the graft, but particularly the PRC layer where the cell count sets a limit on potential visual resolution. Potential survival factors include, but are not limited to, brain-derived neurotrophic factor (BDNF) , fibroblast growth factors (FGFs) , ciliary neurotrophic factor (CNTF) , glial-derived neurotrophic factors (GDNFs) , insulin-like growth factors (IGFs), interleukins (particularly IL-1 alpha, IL-1 beta, and IL-6), neurotrophins (NTs), pigment epithelium-derived factor (PEDF), and anti-apoptotic agents.

The top (innermost, vitread) layer in the graft is the connecting cell layer. The purpose of this layer is to functionally integrate the photoreceptors of the graft with the surviving internuncial or retinal ganglion neurons of the recipient retina. To supply this layer, stem cells, or more committed neural progenitors, can be harvested, expanded, and modified if desired before seeding them into the graft. Prior to transplantation, the cells are removed from proliferation conditions and predisposed to differentiate towards a neuronal fate. Potential sources of stem cells include, but are not limited to, retina-derived neural stem cells and brain-derived neural stem cells. For example, RSCs can be chosen as starting material and a retinal bipolar cell phenotype selected as end point, e.g., using CNTF (Xie and Adler, 2000) (Fig. 10). Other stem cells types that might serve as a starting point for generating linking interneurons include, but are not limited to, embryonic stem cells, mesenchymal stem cells, hematopoietic stem cells, and fat- and skin-derived stem cells. Potential modifications of interest include reporter gene(s), control of cell cycle, control of differentiation, control of migration, control of synaptogenesis, and control of apotosis.

The physical structure of the graft can vary to suit the configuration of the defect but, in general, takes the form of a round or ovoid disc with width ranging between approximately 1.5 mm (clinical macula) and 7 mm (posterior pole) . Other configurations might be advantageous in certain circumstances. The transplantation procedure for the present invention takes advantage of existing vitreoretinal surgical techniques with minimal necessary modifications. The graft of the invention can be delivered to the subretinal space using existing pars plana vitrectomy (PPV) techniques (see Fig. 4) as known in the art. Exemplary PPV techniques are described in Lim et al., 1995, and Ibanez, et al . , 1995. Following preparatory surgery, which might include management of subretinal scarring or CNV, the graft is delivered through a small incision traversing the pars plana 20 using an appropriate instrument such as a soft-tipped cannula 21 or subretinal forceps ( see Fig. 4). Alternatively, a rolled-up graft can be delivered by cannula, in which case the tip of the delivery instrument is directed to a suitable retinotomy (e.g., supero-temporal to the macula) and the rolled-up graft 24 is carefully extruded into the submacular space with care taken to orientation and positioning. The retinal bleb is reduced and the retinotomy is sealed using an air bubble or other potential methods including a bubble of perfluorocarbon gas or using silicon oil (see Fig. 5) . Scleral and conjunctival incisions can be closed with sutures, followed by topical application of anti-inflammatory and antibiotic agents such as dexamethasone and chloramphenicol or gentamicin.

The concept of the invention is further illustrated in Figs. 7A-7C. The graft would consist of the layers described in Figs. 7A and 7B, with the outer retina consisting of the outer nuclear layer 50, containing photoreceptors and the associated pigment epithelium and Bruch 's membrane (obtained from the sources described above) . Attached to the outer nuclear layer 50 would be cells that would provide the linking element (Fig 7B, potential source as described above) , such that a new outer plexiform layer 48 would be formed, integrating the graft with the degenerated inner retinal layers, 46, 44, 42, in Fig. 7C. In Fig. 8, an example of an in vivo practice of the invention is shown. A composite graft consisting of neural stem cells and postnatal photoreceptors was placed into the subretinal space of a rat with a congenic retinal dystrophy. The stem cells are GFP positive (green) , while the transplanted photoreceptors are stained for rhodopsin (red) . In this figure, large areas of integration (yellow) , as well as GFP positive process extension into host retina (green) can be seen, demonstrating that the neural stem cells are capable of providing a crucial linking element between grafted photoreceptors and the degenerating, mature retina.

Other embodiments of the invention may include, inter alia, composite grafts for the clinical repair of other layered neural structures including the cerebral cortex, the cerebellar cortex, the olfactory lobe and the spinal cord. Another use is as a research and development tool to study strategies for neural repair in animal models and in culture, including the evaluation of candidate molecules in culture. The present invention may also be useful for the development of a variety of medical or non- medical biocybernetic systems. These may include, for example, prosthetics or other devices or instruments that incorporate neurons or glia as a means of augmenting or sustaining various forms of information processing, transfer, or storage. For instance, a layered composite graft in which neural connectivity is achieved could serve as a bionic circuit, or chip, that can be linked to other biological or non-biological networks, either through electrical contacts, chemical transmission, or photons. The cells of such devices could be modified and express genes or gene products from other different species. Bionic chips might have advantages forming functional interactions with the wide range of receptors available in living organisms, and could be useful in developing in vitro assay systems for the analysis of potential therapeutic drugs.

The following examples are presented to illustrate the advantages of the present invention and to assist one of ordinary skill in making and using the same. These examples are not intended in any way otherwise to limit the scope of the disclosure. The contents of all cited references (including literature references, issued patents, published patent applications, and co-pending patent applications) cited throughout this application are hereby expressly incorporated by reference.

EXAMPLE I Retinal pigment epithelium-retinal stem cell composite in culture This example shows that a retinal pigment epithelium layer can be transplanted successfully using retinal stem cells as the linking cells. A retinal pigment epithelium (RPE) - retinal stem cell (RSC) composite was made using the following. RPE cells from donor mice were cultured for 7 days (until 50% confluence) in RPE media (10% fetal calf serum in Neurobasal media supplemented with B27) . RSC and RPE cells were then co-cultured for up to 14 days with 10% serum in Neurobasal media supplemented with B27. As shown in Fig. 9, the RPE monolayer preparation (unstained) with retinal stem cells (green) formed a bi-layer composite graft that can be transplanted to the subretinal space of the eye of recipients. EXAMPLE II Generation of bipolar cells from retinal stem cells in vitro

Bipolar cells were generated from retinal stem cells in vi tro for creating bi-laminar composites of the invention for grafting into recipient patients. RSCs were cultured for 14 days in Neurobasal media supplemented with B27, 20 ng/ml of EGF, and 20 ng/ml of ciliary neurotrophic factor (CNTF) . Here, murine retinal stem cells have been treated with CNTF. As shown in Fig. 10, a high percentage of the cells were found to differentiate into bipolar cells based upon morphology as well as expression of the bipolar-specific marker protein kinase C (PKC) . Bipolar cell generation is important to this invention, as this cell type is capable of easily providing the functional neuronal linkage connecting donor photoreceptors with the host inner retinal circuitry.

After 14 days, the cells from Fig. 10 were co-cultured with adult retinal degeneration mouse retina on a tissue culture insert in the same media described above. Explants were grown for 14 days and then examined in thin section. As shown in Fig. 11, grafted cells were developed into bipolar cells, as determined by morphology, laminar localization, and expression of the bipolar specific marker metabotrophic glutaminergic receptor type 6

(mGluRδ; arrows) . In both Figs. 10 and 11, the generation of bipolar cells from retinal stem cells express markers specific for bipolar cells, namely, metabotrophic glutaminergic receptor type 6 (mGluR6) and protein kinase C (PKC) . Newly generated bipolar cell generation is important to this invention, as they are a cell type that can serve as the crucial "linking element", connecting donor photoreceptors with host retina. EXAMPLE III

Transplant of a composite of murine retinal stem cells (RSC) with a biodegradable polymer using pars plana vitrectomy

This experiment shows the successful transplant of an RSC layer supported with a biodegradable polymer. Pig subjects were placed under general anesthesia and prepared and draped in the sterile manner usual to human surgical procedures. The pupil of the left eye was dilated, the conjunctiva was excised, and 3 to 4 sclerotomies were performed 2 mm posterior to the corneal limbus. Ringer's lactate was infused through one of the sclerotomies. Posterior vitrectomy was performed bimanually using an automated vitrector along with a fiberoptic light source, each inserted through the other sclerotomies. The posterior hyaloid was visualized using intraocular fluorescein and removed. A punctate retinotomy was used to access the subretinal space. Cells in suspension were then injected through the punctate retinotomy. Variable degrees of enlargement of the retinotomy were required to introduce cells as neurospheres or when seeded on polymers, and intraocular diathermy was used as needed to maintain hemostasis. The retinotomy was sealed using an air bubble. However, other potential sealing methods include using a bubble of perfluorocarbon gas or using silicon oil. The sclerotomies and conjuntival incision were closed with sutures and topical dexamethasone and chloramphenical were applied. As shown in Fig. 12, an example of how a standard surgical approach, the pars plana vitrectomy (PPV) , can be applied to the invention. Here, an adult pig recipient has undergone vitrectomy and the graft (consisting of a biodegradable polymer and murine retina stem cells) is inserted through a surgical retinotomy into the subretinal space. The biodegradable polymer was made of polylactic and polyglycolic acid (PLA and PLGA, respectively) (supplied by Robert Langer at MIT) . Other polymers may be used such as, e.g., matrigel or hydrogel. The light source and forceps access the vitreous cavity via small incisions through the pars plana.

Fig. 13 shows the result of the above graft 5 weeks after transplantation. The graft is positioned in the subretinal space, without signs of rejection or foreign body response. The retinal stem cells have begun to differentiate into mature neurons.

EXAMPLE IV Graft of murine retinal stem cells without biodegradable polymer

Using the standard pars plana vitrectomy method applied above in Example III, Fig. 14 shows the result of a graft not incorporating biodegradable polymers. Here, a graft of murine retinal stem cells was placed in the subretinal space of an adult pig recipient with an experimentally induced lesion of the RPE layer. The grafted retinal stem cells (green) have formed a monolayer, which restored the integrity of the host RPE cell layer. It is therefore possible that retinal, or other types of stem cells, could be used to generate RPE cells that might: 1) repair damage to the RPE cell layer, such as occurs following the removal of neovascular membranes; 2) generate a new Bruch' s membrane; and 3) form a barrier to neovascular ingrowth from vessels of the choriodal circulation.

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EQUIVALENTS

While the present invention has been described in conjunction with a preferred embodiment, one of ordinary skill, after reading the foregoing specification, will be able to effect various changes, substitutions of equivalents, and other alterations to the compositions and approaches set forth herein.

It is therefore intended that the protection granted by Letters

Patent hereon be limited only by the definitions contained in the appended claims and equivalents thereof.

Claims

CLAIMSWhat is claimed is:

1. A composite graft for treating a patient with a condition associated with photoreceptor loss, comprising: a layer of connecting cells; and a layer of photoreceptors.

2. The graft of claim 1, wherein said connecting cells are selected from the group consisting of neural stem cells, retinal stem cells, brain stem cells, embryonic stem cells and progenitor cells.

3. The graft of claim 1, wherein said connecting cells are capable of generating cells that function as retinal bipolar cells .

4. The graft of claim 1, wherein said connecting cells are capable of forming interneurons that link said photoreceptors to the retina of said patient.

5. The graft of claim 1, wherein said layer of connecting cells are capable of synapsing, following transplantation to said patient, with said layer of photoreceptors of said graft and with surviving retinal interneurons or retinal ganglion cells of said patient .

6. The graft of claim 1, wherein said photoreceptors are cultured from stem cells.

7. The graft of claim 1, wherein said photoreceptors are harvested from a donor.

8. The graft of claim 1, wherein said composite graft further comprises a layer of retinal pigment epithelium.

9. The graft of claim 8, wherein said retinal pigment epithelium is cultured from stem cells.

10. The graft of claim 8, wherein said retinal pigment epithelium is harvested from a donor.

11. The graft of claim 8, wherein said composite graft further comprises a basement membrane to enhance structural integrity of retinal pigment epithelial layer as part of said graft.

12. The graft of claim 11, wherein said membrane is Bruch' s membrane .

13. The graft of claim 12, wherein said Bruch' s membrane is Descemet's membrane, amniotic membrane or lens capsule.

14. The graft of claim 12, wherein said Bruch' s membrane is provided allogeneically, xenogeneically or autologously .

15. The graft of claim 11, wherein said membrane serves as a delivery platform for a bioactive agent.

16. The graft of claim 11, wherein said membrane is a biodegradable polymer.

17. The graft of claim 1, wherein said graft further comprises a bioactive agent for enhancing survival of said graft or for directing phenotypic differentiation.

18. A composite graft for treating a patient with a condition associated with photoreceptor loss, comprising: a layer of photoreceptors; and a bioactive agent for causing bipolar cells of a treated patient to be capable of functioning as connecting cells.

19. The composite graft of claim 18, wherein said bioactive agent is selected from the group consisting of EGF, TGF-alpha, CNTF, NT-3, NGF, BDNF, GDNF, bFGF, sonic hedgehog, IL-1-beta, IL- 6, and TGF-beta.

20. A method of treating a patient with a condition associated with photoreceptor loss, said method comprising the steps of:

(a) providing a composite graft according to claim 1 or claim 18; and (b) transplanting said composite graft to a patient with a condition associated with photoreceptor loss.

21. The method of claim 20, wherein said condition is retinal degeneration.

22. The method of claim 21, wherein said retinal degenerative condition is selected from the group consisting of age-related macular degeneration, cone dystrophy, cone-rod dystrophy, malattia leventinese, Doyne honeycomb dystrophy, Sorsby' s dystrophy, Stargardt disease, pattern/buttefly dystrophies, Best vitelliform dystrophy, North Carolina dystrophy, central areolar choroidal dystrophy, angioid streaks, and toxic maculopathies .

23. The method of claim 20, wherein said condition is retinal detachment, pathologic myopia, retinitis pigmentosa, diabetic retinopathy, CMV retinitis, occlusive retinal vascular disease, retinopathy of prematurity (ROP) , choroidal rupture, ocular histoplasmosis syndrome (POHS) , toxoplasmosis, and Leber' s congenital amaurosis.

24. The method of claim 20, wherein said graft further comprises a layer of retinal pigment epithelium.

25. The method of claim 20, wherein said method further comprises the step of adding a bioactive agent for enhancing survival of said graft or for directing phenotypic differentiation.