Ординатура / Офтальмология / Английские материалы / Retinal Degenerations biology, diagnostics, and therapeutics_Tombran-Tink, Barnstable_2007

adult photoreceptor sheet transplants (72,73). There was no improvement in vision. No clinical signs of rejection were observed, but a subtle, clinically not evident rejection cannot be excluded.

Our group performed retina-only transplants to four patients with RP. In one patient, transient functional improvement was observed as measured by multifocal ERG (mfERG) (74). This patient also reported subjective improvements. However, no changes in Early Treatment Diabetic Retinopathy Study (ETDRS) vision occurred.

Recent Results of First FDA-Approved Clinical Trial: Transplants of Fetal RPE Sheets With Retina Can Improve Vision

The uniqueness of our approach is to co-transplant sheets of human fetal RPE together with its retina. In preclinical experiments, it was shown that in transplants of sheets of human fetal RPE with its retina to athymic nude rats, the RPE sheet can remain as a monolayer of co-transplanted RPE cells with junctional complexes at 9 mo after transplantation, supporting the development and maintenance of transplant photoreceptors (17).

All of our studies met the local Institutional Review Board requirements and state and local laws for the handling of fetal tissue. Our patients’ surgery, preoperative testing, and postoperative testing were funded by a private sponsor to make it possible for the patients to have the surgery without any financial outlay on their part. This private funding has been critical to the success of this clinical trial.

Patient Selection

Our clinical trial focuses on patients with RP and “dry” (non-exudative) AMD, particularly the central areolar choroidal pigment epithelial dystrophy because these diseases have an established pathology and are the most common diseases of the outer retina. Diseases that involve the formation of subretinal neovascular membranes have been excluded, such as presumed ocular histoplasmosis syndrome, and “wet” macular degeneration because of the possible bleeding as well as the excessive trauma to the foveal area that can occur when entering the subretinal space.

In both diseases, the retinal transplantation studies involving both RPE and neural retina in sheets have more potential for measurable success then transplanting either of these tissues alone. To determine the safety of the procedure, we started with patients with the most severe forms of visual problems with RP. As safety was shown, patients in a less advanced stage of the disease with better vision could be selected.

Preoperative Testing and Follow-Up

Complete assessments, ocular examinations, fluorescein angiography, ETDRS protocol for visual acuity testing, photopic mfERG (VERIS science software, EDI Inc., San Mateo, CA), scanning laser ophthalmoscope (SLO), and optical coherent tomography were performed preoperatively and postoperatively and repeated to assess potentially corresponding physiological changes in the region of the transplant. Patients were tested three times preoperatively and at 2 wk and 1, 6, and 12 mo postoperatively. A great deal of consideration was given to what objective preoperative testing should be and could be done to definitively define improvement in vision. The ETDRS visual acuity testing was used as baseline. In addition to this, new techniques needed to be identified that

might be able to show subtle improvements in visual acuity even though the patient might not see any subjective improvement.

The person measuring the ETDRS visual acuity was masked as to which eye had the surgery. The ETDRS protocol for visual acuity testing was followed as described (75). mfERGs and SLO testing were done at different sites in the United States, in the same time frame. The examiners at these sites also did not know which eye had received the transplant. To compensate for the patients’ eye movement after each measurement with the SLO, clearly defined vascular landmarks were used.

Results to Date

As of August 2005, a total of 11 patients received cografts of retina with its RPE, ten with RP and one with “dry” AMD. After FDA approval (BB-IND no. 8354 about clinical retinal transplantation, Principal Investigator Norman D. Radtke), cografted sheets of fetal retina together with its RPE were transplanted in five patients with RP with light perception or no light perception (44). No adverse effects but also no vision improvement were noted. After these five patients, the food and drug administration (FDA) was satisfied with the safety of the procedure, and allowed us to proceed with transplants in patients with vision of 20/800 or worse in one eye. So far, only patients with preoperative vision in the range of 20/800 to hand motion or better have shown improvement.

The most encouraging results have been seen with a patient with RP who was transplanted in February 2002. The preoperative ETDRS vision was 20/800 in the left surgery eye, and 20/400 in the right nonsurgery eye. One year after transplantation, ETDRS vision in the surgery eye improved to 20/160 and remained stable at 20/200 at 2 yr and 2 mo postoperatively whereas the nonsurgery eye remained unchanged at 20/400 (21). Presently, more than 4 yr after transplantation, the vision is still stable. An independent evaluation in another eye hospital by a SLO test showed visual improvement in the surgery eye from 20/270 at 9 mo to 20/84 at 2 yr and 3 mo after surgery. Interestingly, the right eye which had been 20/369 at 9 mo improved to 20/169 at 2 yr and 3 mo (21). However, no changes were observed with mfERG. No clinical evidence of rejection was observed although the transplant sheet lost its pigmentation by 6 mo. The patient’s quality of life has improved so she is now able to read and handle e-mail from a computer, using a magnification glass. She can now write checks, do some sewing, and has taken up her hobby of ceramic painting again. She now predominantly uses her surgery eye, whereas she previously relied on the other unoperated eye. After this patient, the FDA allowed us to use patients with 20/400 vision.

A second patient with RP (surgery in June 2003) showed improvement from hand motion to 20/400. In addition, one patient with AMD (surgery in March 2004) improved from 20/640 to 20/320 at 1.5 yr. With SLO testing at 6 mo, the fixation was over the pigmented transplant area, which means that the patient was using the transplant area. The SLO at 6 mo showed less scatter than preoperatively. Both patients also reported subjective vision improvements. These early findings are encouraging but confirmation from long-term follow-up studies is needed. One RP patient with surgery in March 2003 slightly improved from light perception to hand motion. Another RP patient with surgery February 2004 continued to worsen from 20/400 to 20/300 at 2 yr.

What has been gained from the results to date and where should we proceed? So far, our study has too few patients, so only tentative inferences can be made. The best recipient candidates may be in the range of 20/400 to 20/800 ETDRS vision. Our patient who exhibited the best results had autosomal dominant RP and also received oxygen therapy for 1 wk. Postoperative oxygen treatment is now part of the protocol in all patients on a voluntary basis. The rationale for oxygen treatment is based on studies in experimental animals showing that oxygen reduces the glial reactivity after retinal detachment (76) and preliminary transplantation studies that have shown a beneficial effect of oxygen (77). Another avenue for improvement may be visual training by exposing the patient to visual stimulation patterns, especially during the first 6 months when the grafted cells are developing. This may enforce the use of the transplant for vision and encourage the formation of new appropriate connections.

ANIMAL RESEARCH WITH FETAL SHEET TRANSPLANTS

Retinal Degeneration Animal Models Used for Transplantation

The different retinal degeneration models used for retinal transplantation were extensively reviewed (18,69). For the initial studies (37,38), a light damage model was used in which albino rats were exposed to continuous moderate blue light for 2 to 4 d. This selectively damaged the rod photoreceptors while leaving the RPE intact (78).

Since 1999, we have used three different inherited models of retinal degeneration: albino RCS rats (11,16), and two different lines of transgenic rats with the mutant human rhodopsin S334ter, produced by Chysalis DNX, and kindly provided by M. LaVail, UC San Francisco: the fast degenerating line 3 (79), and the slow degenerating line 5 (80).

Morphological Repair of Damaged Retinas by Fetal Retinal Sheets

With and Without RPE

The following sections will present the results from research performed to transplant fetal retinal sheets with and without its RPE in animal models of retinal degeneration, using our custom-made implantation instrument and procedure which gently places the tissue into the subretinal space (reviewed in refs. 18,69; see Technical Challenges With Fetal Sheet Transplantation section).

Only 20 to 30% of fetal sheet transplant in rodents are laminated similar to a normal retina with photoreceptor OS in contact with grafted (16) or host RPE (37,81). The rest of the transplants will develop rosettes sometimes with parallel inner retinal layers. The crucial problem is to insert the instrument nozzle at the correct angle to place the tissue into the subretinal space without damaging the host RPE or Bruch’s membrane, or placing the tissue into the vitreous. This is very delicate in rodents because the transscleral approach does not allow the surgeon to see the operation area in the eye.

There is also a “time window” for successful transplantation because the retina and the RPE seal together in later stages of retinal degeneration, and this glial seal prevents the insertion of anything into the subretinal space.

The preparation of cografts poses an additional challenge: because fetal retina has not yet developed OS, the RPE can easily detach from its retinal sheet, and it requires very careful dissection to keep both together. An example of a human RPE cografted with retina

Fig. 1. Human fetal pigmented RPE cografted with its neural retina in subretinal space of athymic albino nude rat, in contact with photoreceptor outer segments of transplant photoreceptors. Part of Bruch’s membrane and the choroid can be seen in the right lower corner of the micrograph. Donor 14 wk, 8.9 mo after transplantation. Bar = 5 µm. OS, outer segment; RPE, retinal pigment epithelial.

to an athymic albino nude rat is shown in Fig. 1; a rat fetal cograft to a S334ter line 5 rat is shown in Fig. 2A–D.

Connectivity

The integration of retinal aggregate transplants appears to depend on the status of host photoreceptors (82). If the host photoreceptors are lost completely, bridging neuronal fibers can be seen between transplants and hosts. Similar results can be seen with fetal retinal sheet transplants in retinal degenerate rats. Connectivity and transplant “integration” is also related to the reaction of glial cells. It has recently been shown that retinal aggregate transplants integrate better in transgenic mice that lack both of the intermediate filaments vimentin and glial fibrillary acidic protein (83).

Both the fetal retinal sheet transplant and the host have an inner nuclear layer with various retinal interneurons. If transplant and host neurons form synaptic connections, the wiring would be different from a normal retina. It is still unknown to what extent the host retina and brain can extract information from light stimulation of the transplant. However, our results indicate that the transplants have a direct effect on visual responses (see Section Visual Function of Transplants Demonstrated by Electrophysiology), and that transplants form synaptic connections, albeit to a limited extent, with the host retina (see next Section Indications for Transplant-Host Connectivity).

Indications for Transplant-Host Connectivity

Sometimes, transplants are too perfect and form a glial barrier towards the host (e.g., Fig. 2 in ref. 69). In other cases, the transplants appear to merge with the host retina so that it is sometimes impossible to tell where the transplant ends and where the host retina begins. Examples for this are shown in Fig. 2. The current work is focused on

Fig. 3. Transsynaptic virus tracing—an indication for synaptic connections. Ba Blu virus expressing Escherichia coli β-galactosidase was injected into the visual brain center, the superior colliculus (SC), and detected by X-gal histochemistry (A–D), and electron microscopy (E,F) in the retina. (A,B,E) Normal retina; (C,D,F) transplants. (A,B) Normal retina, 48 h (A) and 67 h (B) after virus injection into the SC. (A) Müller cells are labeled with one ganglion cell. (B) With longer survival times, the virus is spreading through all retinal layers and also laterally. (C,D) Transplant to s334ter transgenic rat, 65 h after Bablu virus injection into SC. Arrows point to some virus-labeled cells in transplant with apparent neuronal morphology. E18 retinal transplant, 9.8 mo after transplantation. (E) Normal retina at 2 d (52 h) after virus injection: virus-labeled ganglion cell. Arrows point to enveloped virus. (F) Cograft of retina with RPE (E20) to RCS rat, 68 h after Bablu virus injection into SC, and 4.9 mo after transplantation. Arrowheads indicate nonenveloped virus in nucleus and cytoplasm. Arrows indicate enveloped virus in cytoplasm. Magnification bars: A–D: 20 µm; E,F: 1 µm. H, host; T, transplant; GC, ganglion cell layer; IP, inner plexiform layer; IN, inner nuclear layer; OP, outer plexiform layer; ON, outer nuclear layer; OS, outer segments; PRV, pseudorabies virus.

attenuated pseudorabies virus (PRV), which is specifically transferred from one neuron to the next at synaptic contact points, and transported exclusively in the retrograde direction (84). This tracer, PRV Bartha, has been used for outlining multisynaptic circuitry in the CNS for many years (85). The transfer of the virus between neurons depends on the development of functional synapses (86). A somewhat confusing issue for scientists unfamiliar with the virus properties is that the virus can infect glial cells. Glial cells can however not produce a complete infectious virus so that no virus can leave the cell. This prevents nonspecific contamination (84). Neuronal cells are labeled in retinal sheet transplants after injection of PRV into the superior colliculus into an area corresponding to the placement of the transplant in the retina (19). Examples are shown in Fig. 3. However, Müller cells of the host retina inevitably are labeled shortly

after label of the ganglion cells, but cannot produce infectious enveloped virus, similar to what has been shown in the brain.

Retinal Sheet Transplants Restore Visual Function

Light/Dark Shift of Phototransduction Proteins in Transplant Photoreceptors

Are transplant photoreceptors capable of responding to light?

Certain phototransduction proteins, such as arrestin (S-antigen) and rod α-transducin, are found in different compartments of the rod photoreceptor dependent on the light/dark cycle (87). Arrestin is in the inner segments in the dark and in the OS in the light, whereas the distribution for rod α-transducin is reversed. The cytoskeleton is required for the translocation of these proteins (88). The translocation of arrestin does not depend on transducin signaling (89).

Photoreceptors in sheet transplant to light-damaged rats show translocation of arrestin and rod α-transducin dependent on the light cycle, indicating that the phototransduction process takes place and that they respond to light (38). This was shown by fixation of transplanted rat eyes either in light or dark.

Visual Function of Transplants Demonstrated by Electrophysiology

By recording from the primary target of retinal ganglion cells in rodents, the superior colliculus (SC), we showed in several rodent models of retinal degeneration that visually evoked multi-unit responses could be recorded in a small area of the SC corresponding to the retinal placement of the transplant (11,79–81). The visual stimulus consisted of a bright light flash that specifically stimulated cone photoreceptors. A common phenomenon with all responses from transplanted rats was the longer latency (i.e., the response time) as compared with normal control rats.

In albino RCS rats, responses were found in 19 of 29 transplanted rats. Visual responses were also found in age-matched sham surgery rats; however, the responses from transplanted rats had higher amplitudes and shorter latencies (11). The rats were transplanted at the age of 37 to 69 d, when photoreceptors are already committed to apoptosis, and it would be unlikely for transplants of RPE only to have a rescue effect at this age on host photoreceptors as shown previously (90).

In transgenic S334ter-3 rats with rapid retinal degeneration, responses were found in 7 of 11 transplanted rats, but not in any sham surgery rats (79). In transgenic S334ter line-5 rats with slow retinal degeneration, transplant-derived responses could be distinguished in 8 of 13 rats at 8 mo of age when a scotoma had developed in the transplant area (80). In all three rat models, no rescue effect of the transplant on host cones was seen with qualitative immunohistochemistry for S-antigen (arrestin) which labels both rods and blue cones. The quality of responses appeared to depend on the quality of the transplant (better laminated transplants gave better responses). If a rescue effect was the case, disorganized transplants should also have given a response.

This was different in rd mice, where responses were found in three of seven mice transplanted at the age of 31 to 38 d (81). The three light-responsive eyes had rosetted or disorganized grafts, but no responses were found in eyes with well-laminated grafts. On the other hand, the immunoreactivity for recoverin in the host retina overlying the graft was much higher in the responsive eyes compared with untreated age-matched

rd/rd mice, or nonresponsive grafted eyes. Thus, in rd mice, the restoration of visual responses by retinal sheet transplants appeared to depend on a rescue of host cones. A previous study with retinal aggregate transplants found ganglion cell responses in 3 of 10 mice transplanted at the age of 2 wk, but no responses in mice transplanted at an older age (91). This was in contrast to the recent study which showed an effect in mice transplanted at the age of 30–38 d (81), demonstrating the advantage of sheet transplantation.

Transplant Effect on Visual Acuity

Optokinetic head tracking has been used to test visual acuity in pigmented rats to show the time course of retinal degeneration in different models (92–94) and photoreceptor rescue after RPE or Schwann cell transplantation (92,95,96). Using an improved design which made it possible to test each eye independently, it could be demonstrated that the deterioration of the optokinetic response in S334ter-3 rats with rapid retinal degeneration could be delayed by retinal sheet transplants (97).

Mechanism of Transplant Effect on Vision

Two mechanisms appear to be involved: a trophic effect by the transplanted fetal tissue, resulting in rescue of host cones (in refs. 20,98 for reviews); and a direct effect from transplant photoreceptors mediated by synaptic connections between transplant and host (19). So far, visual responses from transplanted animals in the SC could only be recorded at mesopic or photopic light levels where cones can be stimulated, and not at scotopic light levels less than –3 log cd/m2. Normal rats show responses less than –5 log cd/m2 (99). The mechanism of visual restoration appears to be different in rd mice (because there was no correlation between transplant organization and visual responses) (81) and the different rat degeneration models where a correlation between transplant organization and visual responses was found (11,79,80). Irrespective of the mechanism involved, our results clearly show that transplants have a beneficial effect on vision.

SUMMARY OF RESEARCH ACCOMPLISHMENTS BUILDING THE BASIS FOR CLINICAL TRIALS

The research presented in this chapter has demonstrated many accomplishments indicating the viability of retinal transplantation as a remedy for retinal diseases. These include:

1.Use of a gentle procedure and instrument.

2.Maintenance of the primordial circuitry between the donor retinal neuroblastic cells by sheet transplantation.

3.Reconstruction of an area of a damaged retina by a laminated transplant resembling a normal retina.

4.Ability of transplant photoreceptors to transform light into electrical signals as normal photoreceptors, as indicated by light/dark shift of phototransduction proteins.

5.Synaptic connections between transplant and host retina are indicated by transsynaptic tracing from the host brain.

6.Light sensitivity can be restored or preserved in brain area that corresponds to the placement of the transplant in the retina.

7.Retinal degeneration rats can preserve functional vision after transplantation in a visual acuity optokinetic test.

8.Many retinal diseases require the replacement of both photoreceptors and RPE. Fetal retinal epithelium can be gently co-transplanted with the retina and remain as a monolayer sheet after ten months, apparently supporting exchange of nutrition between the choroid and the photoreceptors.

9.The donor tissue is well tolerated in the subretinal space, can survive without immunosuppression, and is safe to use in patients.

10.In several retinal degeneration models, intact-sheet transplants unequivocally have beneficial effects on vision, as shown also in recent clinical trials.

FUTURE DIRECTIONS

There are indications that synaptic connections between host and graft are involved in the beneficial effects from retinal transplantation. Although now only limited connections may be involved, the positive sign is that there might be a large margin for improvement. The increase of appropriate graft-host connections might be helped with several different treatments: (1) Treatment with brain-derived neurotrophic factor has shown promising effects in preliminary studies, as well as introduction of other trophic factors. (2) Use of a laser to shave off the superficial layer of the donor tissue may improve integration. (3) A noninvasive treatment with “visual stimulation” of patients in the first 6 mo after surgery when the immature graft cells are developing has a good probability to improve results.

After many years of dedicated research, retinal transplantation appears very promising as a viable treatment to bring hope to patients with “incurable” retinal diseases.

ACKNOWLEDGMENTS

This work was supported by funds from an anonymous sponsor; The Vitreoretinal Research Foundation, Louisville KY; The Foundation Fighting Blindness; the Murray Foundation Inc., New York; an unrestricted grant from the Research to Prevent Blindness; NIH EY08519; NIH EY03040; Foundation for Retinal Research; and Fletcher Jones Foundation.

The authors want to thank Zhenhai Chen, Xiaoji Xu, Betty Nunn, and Lilibeth Lanceta for their technical assistance.

The authors can acknowledge only the most significant collaborators involved owing to space limitations. Biju Thomas, Guanting Qiu, Srinivas Sadda (Doheny Eye Institute, Los Angeles, CA); Shinichi Arai (now Niigata University, Niigata, Japan); Botir T. Sagdullaev (now Washington University, St. Louis); Norman D. Radtke, Heywood M. Petry, Maureen A. McCall; Peng Yang (University of Louisville); Gustaw Woch (Hershey Medical School, Hershey, PA); and Sherry L. Ball (Cleveland University, Cleveland, OH) contributed to the work presented in this chapter. We thank J.P. Card, University of Pittsburg, and Lynn Enquist, Princeton University, for their gift of the pseudorabies virus strains, and their helpful advice; Matthew M. LaVail, UCSF, for the founder breeding pairs of transgenic S334ter rats; and Eric Sandgren, University of Wisconsin, for founder breeders of transgenic hPAP rats.

Robert B. Aramant, Norman D. Radtke, and Magdalene J. Seiler have a proprietary interest in the implantation instrument and method.

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