WO1994021204A1

WO1994021204A1 - Method for rescuing endogenous cone photoreceptor population

Info

Publication number: WO1994021204A1
Application number: PCT/US1993/008567
Authority: WO
Inventors: Martin S. Silverman
Original assignee: Photogenesis, Incorporated
Priority date: 1993-03-16
Filing date: 1993-09-10
Publication date: 1994-09-29
Also published as: EP0706357A1; CA2158442A1; AU4856993A; IL107496A0

Abstract

Method for rescuing the endogenous cones of the eye of an individual afflicted with an inherited or acquired disease which is causing a dysfunction of the individual's endogenous rods. In the method, a rod-containing graft is transplanted to a position in the individual's eye from which the transplanted rods can exert a trophic influence upon the endogenous cones. Preferably the rod-containing graft has the same organization and cellular polarity as is present in a normal outer nuclear layer.

Description

METHOD FOR RESCUING ENDOGENOUS CONE PHOTORECEPTOR POPULATION

BACKGROUND OF THE INVENTION

The present invention is directed to a method for rescuing the endogenous cone photoreceptor population of persons afflicted with an inherited or acquired human retinal disease in which the rod photoreceptors are defective, thereby leading to the progressive loss or dysfunction of rod photoreceptor cells which in turn leads to cone photoreceptor dysfunction, dystrophy and/or loss.

Recent demonstrations of survival-promoting activity by neurotrophic agents in diverse neuronal systems have raised the possibility of pharmacological therapy for inherited and degenerative disorders of the central nervous system, in general, and the retina, in particular. The retina is the sensory epithelial surface that lines the posterior aspect of the eye, receives the image formed by the lens, transduces this image into neural signals and conveys this information to the brain by the optic nerve. The retina comprises a number of layers, principally, the ganglion cell layer, inner plexiform layer, inner nuclear layer, outer plexiform layer, outer nuclear layer, photoreceptor inner segments and outer segments. The outer nuclear layer comprises the cell bodies of the photoreceptor cells with the inner and outer segments being extensions of the cell bodies.

The photoreceptor population is composed of two subclasses of photoreceptor cells, known as rods and cones. Cones function during relatively high luminance levels and subserve high acuity day or photopic vision as well as color vision. Cone cell density peaks at the center of the retinal fovea (known as the foveola) and then drops off rapidly as a function of distance from the fovea. In contrast, rods function during relatively low light levels and subserve scotopic or night vision of relatively low acuity. Rods are absent from the center of the human fovea, first appearing at distances of 100-200 μm from the foveal center. The rod distribution is more uniform across the retina as a function of eccentricity except for a central region. While no rods are present in the foveola, the density of rods increases sharply outside the fovea with the highest rod densities being found in a broad, horizontally oriented elliptical ring at approximately the same eccentricity as the center of the optic disk. Curcio et al., "Human Photoreceptor Topography", The Journal of Comparative Neurology, 292:497-523 (1990).

Several forms of blindness are primarily related to the progressive loss of photoreceptor cells caused by diseases in select populations of cells. Such diseases include Retinitis P gmentosa, age-related macular degeneration and at least certain bulls-eye retinopothies.

Retinitis pigmentosa is the name given to a group of inherited disorders which give rise to degeneration of the outer nuclear layer of the retina; in each of these disorders, a genetic defect leads initially to the progressive loss of rod photoreceptors and subsequently to eventual cone photoreceptor dysfunction, dystrophy and/or loss. Age-related macular degeneration is the leading cause of visual loss in the elderly in many developed countries. The topography of chorio-retinal abnormalities associated with incipient age-related macular degeneration shows that the annular rod-rich ring surrounding the fovea is first affected and the disease then progresses to the fovea.

Bulls-eye retinopothy may result from chemical toxicities such as chronic exposure to chloroquinone and, in addition, may develop from presently unknown genetic abnormalities. Like age-related macular degeneration, the topography of damage associated with bulls-eye retinopothy shows that the annular rod-rich ring surrounding the fovea is first affected and the disease then progresses to the fovea. Faktorovich et al., Nature (London) 34_7: 83-86 (1990) explored the survival-promoting activity of basic fibroblast growth factor (bFGF) on degenerating photoreceptor cells in the RCS rat with inherited retinal dystrophy. It was reported that intravitreally and subretinally injected bFGF delayed photoreceptor degeneration in this inherited disorder. However, injection of bFGF is not without its side-effects; injection can result in an increased incidence of retinal macrophages, retinal fibrosis, retinal neovascularization and cataracts. In addition, injection only delayed the degeneration; it did not avoid it. Moreover, unlike the primate, the outer nuclear layer of the rat is composed of 97% of rods and, therefore, rescue was primarily provided for genetically normal, albeit traumatized, rod photoreceptors. Furthermore, there is no instance of human photoreceptor dystrophy that is presently known to be caused by the genetic defect seen in the RCS rat (RPE deficit in outer segment phagocytosis). Thus, this model does not appear to be a relevant model for Retinitis Pigmentosa, age-related macular degeneration, bulls-eye retinopothy and other such disorders. Similarly, La Vail et al., Proc. Natl. Acad. Sci.

USA, 82:11249-11253 (1992) disclose that intraocular administration of various trophic factors to a light damaged animal can rescue the photoreceptor layer. Once again, however, this animal model does not appear to be highly relevant to human diseases; the authors conclude the paper by acknowledging that "[t]he ultimate therapeutic potential of each agent for use in any inherited or acquired human retinal disease remains to be determined.... Most importantly, it will be necessary to evaluate the ability of each agent to rescue photoreceptors or other retinal neurons in animal models that are directly relevant to human retinal disease."

SUMMARY OF THE INVENTION Among the objects of the present invention, therefore, may be noted the provision of a method for rescuing the endogenous cones of an individual afflicted with an inherited or acquired retinal disease which causes a progressive loss of rods and subsequent eventual cone dystrophy, dysfunction and/or loss, the provision of such a method which does not require repeated invasive procedures to maintain the cones, and the provision of such a method which does not result in an increased incidence of retinal macrophages, cataracts and other abnormalities. Briefly, therefore, the present invention is directed to a method for rescuing the endogenous cones, the method comprising transplanting a rod-rich graft to the individual's eye after it has been determined that he or she is afflicted with an inherited or acquired retinal disease which has caused or is causing a loss of endogenous rods. The transplant is performed at a time when the individual still possesses cone cells.

Other objects of the invention will be in part apparent and in part pointed out hereinafter.

BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1 is a schematic of a donor retina;

Fig. 2 is a schematic of a flattened retina;

Fig. 3 is a schematic of a flattened retina mounted to a substrate; Fig. 4 is a schematic of a sectioned retina mounted to a substrate;

SUBSTITUTE SHEET Fig. 5 is a schematic of a laminate comprising a retina section on a supporting, stabilizing substrate;

Fig. 6 is a schematic top plan view of the laminate of Fig. 7, showing a graft (dashed lines) comprising a photoreceptor cell layer and a supporting, stabilizing substrate;

Fig. 7 is a schematic of the laminate mounted on a plate formed with spacers;

Fig. 8 is a schematic of the laminate mounted on a plate infused with molten gelatin with a cover plate;

Fig. 9 is a schematic of the graft being skived;

Fig. 10 is a horizontal section through an eye illustrating a pars plana surgical approach with the surgical instrument inserted into a bleb; Fig. 11 is a photograph (200X original magnification) of a plastic embedded section of the retina of a rd mouse sacrificed at 21 days of age;

Fig. 12 is a photograph (200X original magnification) of a plastic embedded section of the retina of a rd mouse sacrificed at 3 months of age;

Figs. 13 and 14 are photographs (200X original magnification) of plastic embedded sections of the retinas of two rd mice sacrificed at 3 months of age, each of the animals having received a rod-rich transplant at 21 days of age to the superior retina;

Figs. 15 and 16 are photographs (600X original magnification) of plastic embedded sections of the retinas of two rd mice sacrificed at 3 months of age, each of the animals having received a rod-rich transplant at 21 days of age to the superior retina;

Fig. 17 and 18 are photographs (200X original magnification) of plastic embedded sections of the retinas of two rd mice sacrificed at 3 months of age, each of the animals having received a rod-rich transplant at 21 days of age to the superior retina; and

Figs 19 and 20 are photographs ( 200X original magnification) of plastic embedded sections of the retinas of two rd mice sacrificed at 3 months of age, each of the animals having been subjected to a sham operation at 21 days of age to the superior retina.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Surprisingly, it has been discovered that cones gradually and progressively become dysfunctional in the absence of rods and that endogenous cones can be rescued by rod- containing grafts transplanted after a near total loss of endogenous rod cells. Accordingly, rod-containing grafts transplanted to the subretinal space of the eye will maintain the endogenous cone populations in individuals afflicted with an inherited or acquired disease which causes rod dysfunction or disease after the endogenous rod population has become diseased or is otherwise dysfunctional. Such diseases may include Retinitis Pigmentosa, age-related macular degeneration and various bulls-eye retinopothies.

To conserve a maximum of photopic vision, therefore, the rod-containing graft is preferably transplanted to the eye as soon as possible after it is determined that the host is afflicted with a disease which has caused or is causing a rod dysfunction and that a progressive visual loss will follow. For those afflicted with Retinitis Pigmentosa, it is preferred that the rod-containing graft be transplanted as soon as significant scotopic vision is lost and it is determined that visual loss will be progressive. Similarly, for those afflicted with age-related macular dystrophy the transplant will be made after the host manifests a central regional decrease of scotopic vision or develops an abnormal appearance

SUBSTITUTE SHEET in his or her fundus such as pigmentary changes, the formation of heavy Drϋsen deposits, and the loss of the foveal reflex. For those afflicted with chemical toxicity (or other bulls-eye retinopothy), the graft is preferably transplanted after the occurrence of a significant visual defect which is progressive and likely to significantly affect photopic vision. In all instances, however, the rod-containing graft should be transplanted before there is a complete loss of endogenous cones. The graft should be transplanted to a position which allows it to exert a trophic influence upon the endogenous cones and recent experimental evidence suggests that the rod- containing graft need not be in apposition to the endogenous cones to maintain them. Nevertheless, it is presently preferred that at least a portion of the graft be located in or near what is or what was the host's endogenous rod-containing ring; that is, a portion of the graft is preferably located within about 5 millimeters, more preferably about 3 millimeters of the foveal center. To avoid possible disruption of high- acuity vision, however, it is additionally preferred that the graft be located outside the fovea, and more preferably at least about 2 millimeters from the foveal center.

The rod-containing graft may be transplanted in various shapes and sizes depending upon the diseased state. For instance, the graft may be an annular ring having an inner diameter of about 2 millimeters and an outer diameter of at least about 6 millimeters with a radial incision extending from the inner to the outer diameter; such a graft can surround the fovea and subtend a substantial fraction of the macular region and be transplanted without disruption of the fovea. Alternatively, 2 or more grafts each having a triangular, rectangular, trapezoidal or other desired shape can be transplanted. The rod-containing graft is preferably rich in rods. That is, it preferably comprises predominantly rods and few cones. Although the rod-containing graft may comprise the entire thickness of a donor retina (i.e., the ganglion cell layer, inner plexiform layer, inner nuclear layer, outer plexiform layer, outer nuclear layer and photoreceptor inner and outer segments), it is preferred that the rod-containing graft be a section of a donor retina having the inner retinal layers removed and the photoreceptors exposed, that is, it is preferred that the rod-containing graft contain the outer nuclear layer and the associated photoreceptor processes (inner and outer segments ) .

Referring now to Fig. 1, a rod photoreceptor rich

_* graft is prepared by removing a donor retina 50 comprising inner retina layers 52 and photoreceptor layer 54 from a donor eye. The donor retina 50 is flattened (Fig. 2) by making a plurality of cuts through the retina from locations near the center of the retina to the outer edges thereof (see Fig. 6). Cuts can be made in other directions if necessary. As shown in Fig. 3, the flattened retina 56 is placed with the photoreceptor side 54 down on a gelatin slab 58 which has been surfaced so as to provide a flat surface 60 that is parallel to the blade of a vibratome apparatus. The gelatin slab 58 is secured to a conventional vibratome chuck of the vibratome apparatus. Molten four to five percent gelatin solution is deposited adjacent the flattened retina/gelatin surface interface 61 and is drawn by capillary action under the flattened retina causing the flattened retina to float upon the gelatin slab 58. Excess molten gelatin is promptly removed and the floating flattened retina is then cooled to approximately 4 °C with ice-cold Ringer's solution that surrounds the gelatin block to cause the molten gelatin to gel and thereby coat the

SUBSTITUTE SHEET bottom surface of the flattened retina and adhere it to the gelatin block.

As shown in Fig. 4, the inner retina portion 52 is sectioned from the top down at approximately 20 to 50 millicrons until the photoreceptor layer 54 is reached, thereby isolating the photoreceptor layer from the inner layers of the retina. The vibratome stage is advanced and a section from approximately 200 to 300 millimicrons thick is obtained as shown in Fig. 5. The thickness of this section is sufficient to undercut the photoreceptor and form a laminate 62 consisting of a layer of photoreceptor cells and the gelatin adhered thereto.

As shown in Fig. 6, the laminate 62 is cut vertically along the dashed lines to create a graft 63 having a size appropriate for transplantation.

Gelatin is presently preferred as a substrate and as the adhesive to laminate the retinal tissue to the substrate because of its flexibility, apparent lack of toxicity to neural tissue and ability to dissolve at body temperature. However, other compositions may be substituted as the substrate and other compositions such as concanavalin A, wheat germ agglutin, or photo reactive agents which gel or solidify upon exposure to light and which also have the desirable characteristics of gelatin may be substituted as the adhesive. Advantageously, the gelatin or other substrate may additionally serve as a carrier for any of a number of trophic factors such as fibroblast growth factor, pharmacologic agents including immunosuppressants such as cyclosporin A, anti- inflammation agents such as dexamethasone, anti-angiogenic factors, anti-glial agents, and anti-mitotic factors. Upon dissolution of the substrate, the factor or agent becomes available to impart the desired effect upon the surrounding tissue. The dosage can be determined by established experimental techniques. The substrate may contain biodegradable polymers to act as slow release agents for pharmacologic substances that may be included in the substrate. In addition, the laminate may be treated prior to transplantation with these factors to increase viability of the transplant.

As an alternative to mechanical, e.g., microtome sectioning, the donor retina may be chemically sectioned. Specifically it is known that neurotoxic agents such as kainic acid and anoxia are toxic to cells in all retinal layers except the photoreceptor layer. Thus, if the donor retina is treated with an appropriate neurotoxic agent the photoreceptor layer can be isolated. This technique has the advantage of maintaining the retinal Muller cells (which are relatively insensitive to kainic acid and anoxia) with the photoreceptor cells.

The laminate 63 may be transplanted as-is using a surgical instrument as described in copending application Serial No. 07/566,996 (which is incorporated herein by reference). To permit the transplantation of relatively large grafts through relatively small incisions, however, it is preferred that the graft be further prepared and transplanted as further described herein.

As shown in Fig. 7, prior to transplantation the laminate 63 is placed onto a flat plate 64 formed with risers 66. The plate 64, with the laminate 63 positioned between the risers 66 is then infused with molten fifteen to twenty percent gelatin solution. The laminate 63 is surrounded and covered by the molten gelatin. A flat cover plate 68 (Fig. 8) is placed on top of the risers 66 to remove any excess molten gelatin and to establish the precise thickness of the graft.

After the molten gelatin is allowed to gel, cover plate 68 and risers 66 are removed to expose gelatin slab 70.

10

SUBSTITUTE SHEET The gelatin slab 70 is cut to a proper size for transplantation and opposite sides 72 of the gelatin slab are 'skived--cut at obtuse and acute angles relative to the top and bottom surfaces of the gelatin slab to produce a graft 74 (Fig. 9) having approximately parallel sides. Using a funnel and a viscoelastic fluid, graft 74 is coiled to form a volute 76 (Fig 10); the skived sides readily slip relative to each other without causing damage to the photoreceptors.

The host eye is prepared so as to reduce bleeding and surgical trauma. A scleral pars plana approach to the subretinal space is preferred (Fig. 10), but other approaches may be used. A small incision is made in the pars plana large enough to insert the surgical instrument 78 (about 0.75 mm - about 2.0 mm). Following vitrectomy, the eye is cooled by infusion of cooled balanced salt solution through a second pars plana port into the vitreal cavity of the eye (not shown), to avoid dissolution of the volute 76 during the surgical procedure. A portion of the retina 82 at the site of transplantation is raised away from the pigment epithelial cell lining 84 by making an incision in the retina and infusing balanced salt solution in the subretinal area to form a bleb 80 at the transplantation site of the retina 82. The instrument 78 is inserted through the pars plana pert, the vitreal cavity and into the subretinal space. The entire tip of the instrument 78 is inserted in the bleb 80 and the volute 76 is ejected by carefully advancing a plunger 86. The ejected volute 76 uncoils under its inherent uncoiling memory as it is ejected. If the volute 76 does not uncoil entirely, micro picks can be used to completely uncoil it. The bleb 80 is then deflated so that the graft is held in a sandwich-like arrangement at the desired position by the retina 82 and pigment epithelial cell lining 84. The gelatin encapsulant dissolves once the eye returns to normal body temperature.

11 The following examples illustrate the invention.

EXAMPLE 1

A series of experiments were performed using the rd mouse, an animal in which the rod photoreceptor expresses a genetic abnormality which leads to the death of the rod cells. At about 10 days of age (as in the normal mouse), the rd mouse contains about 10 layers of photoreceptor cells. By 21 days (Fig. 11), only one layer of photoreceptors remain which are almost exclusively cones; the cones that remain tend to have a fusiform and shrunken appearance with few if any outer processes. By 3 months of age (Fig. 12) essentially all photoreceptors have been lost.

At 21 days of age, viable, non-dystrophic rod-rich grafts prepared as described above which were harvested from a neonatal congenic control animal (i.e., a mouse which has all the genetic characteristics of the rd mouse but which does not express the rd trait) were transplanted to a relatively small area within the superior subretinal space of rd mice. The animals were sacrificed at 3 months of age and plastic embedded sections were prepared. The results are depicted in Figs 13- 18.

Figs. 13 and 14 show that the monolayer of cones survived in animals receiving the rod-containing grafts. As indicated by Fig. 12, by this age essentially all photoreceptors should have been gone.

Figs. 15 and 16 show that the effect of the transplant is regional, that is, areas adjacent to the transplant tend to show a much greater rescue phenomena. The superior retina (the site of the transplant) has a monolayer of endogenous cones that are retained whereas in the inferior retina (below the optic nerve and away from the transplant) nearly all cones are gone. Also, the rescued endogenous cones

12

SUBSTITUT E SHfcET appear more healthy than is typical for photoreceptors of even

21 day-old animals.

Figs. 17 and 18 also show rescued endogenous cones which have hypertrophied, appear round, and some have extended processes, thus showing a reestablishment of some of the morphological differentiation that is characteristic of the cone photoreceptor.

To eliminate the possibility that the rescue effect demonstrated by Figs 13-18 is due to surgical trauma, sham operations were performed on rd mice at 21 days of age wherein the identical surgical procedure was performed except that no graft was transplanted. At 3 months of age, these animals were sacrificed and plastic embedded sections were taken and photographed. As shown in Figs. 19 and 20, the sham operation causes no rescue effect in that there is an essential total loss of endogenous in both the superior and inferior retina. In view of the above, it will be seen that the several objects of the invention are achieved and other advantages attained. As various changes could be made in the above method without departing from the scope of the invention, it is intended that all matter contained in the above-description or shown in the accompanying drawings be interpreted as illustrative and not in a limiting sense.

13

Claims

WHAT IS CLAIMED

1. A method for rescuing the endogenous cones of the eye of an individual afflicted with an inherited or acquired disease which has caused or is causing rod dysfunction, the method comprising transplanting a rod-containing graft to the individual's eye which will maintain the endogenous cones.

2. The method of claim 1 wherein the rod-containing graft has the same organization and cellular polarity as is present in normal outer nuclear layers.

3. The method of claim 1 wherein the disease is inherited.

4. The method of claim 1 wherein the transplant is made after the individual manifests a loss of scotopic vision or develops an abnormal appearance in his or her fundus.

5. The method of claim 1 wherein the transplanted rod-containing graft is not in apposition to the endogenous cones.

6. The method of claim 1 wherein at least a portion of the transplanted graft is located in what is or what was the individual's endogenous rod-containing ring.

7. The method of claim 1 wherein a portion of the transplanted graft is located within about 5 millimeters of the foveal center.

8. The method of claim 1 wherein a portion of the transplanted graft is located within about 3 millimeters of the foveal center.

14

SUBSTITUTE SHEET

9. The method of claim 1 wherein the transplanted graft is located within about 5 millimeters of the foveal center but at least 2 millimeters outside the foveal center.

10. The method of claim 1 wherein the graft comprises a photoreceptor layer and gelatin.

11. A method for rescuing the endogenous cones of the eye of an individual afflicted with an inherited or acquired disease which is causing a dysfunction of the individual's endogenous rods, the method comprising transplanting a rod-containing graft to an area of the individual's eye from which the transplanted rods can exert a trophic influence upon the endogenous cones, the rod-containing graft having the same organization and cellular polarity as is present in normal outer nuclear layers.

12. The method of claim 11 wherein the transplanted rod-containing graft is not in apposition to the endogenous cones.

13. The method of claim 11 wherein a portion of the transplanted graft is located within about 5 millimeters of the foveal center.

14. The method of claim 11 wherein a portion of the transplanted graft is located within about 3 millimeters of the foveal center.

15. The method of claim 11 wherein the transplanted graft is located within about 5 millimeters of the foveal center but at least 2 millimeters outside the foveal center.

15

SUBSTITUTESHEET

16. The method of claim 11 wherein the graft comprises a photoreceptor layer and gelatin.

17. The method of claim 11 wherein the graft is transplanted to the subretinal space of the individual's eye.

16

SUBSTITUTE SHE