Vision Change After Sheet Transplant of Fetal Retina With Retinal PigmentEpithelium to a Patient With Retinitis Pigmentosa FREE

Previously, it has been reported that intact sheets of fetal retinatogether with its retinal pigment epithelium (RPE) can be safely transplantedto patients with retinitis pigmentosa (RP).¹ Retinitispigmentosa is a group of inherited diseases with mutations in photoreceptoror RPE genes.² In these diseases, blindnessis due to specific degeneration of the photoreceptors and/or RPE cells eventhough the inner retina that connects to the brain may still remain functional.^{3 - 5} If the diseased photoreceptorsand/or RPE can be replaced and the new cells make appropriate connectionsto the functional part of the host retina, eyesight may be improved.

Using an implantation instrument and procedure originally developedby Aramant and Seiler⁶ for use in rats withretinal degeneration, clinical studies have been performed in patients withRP^{1 ,7} to transplant intact sheetsof fetal retina with or without the RPE. Although no objective improvementin vision was reported, these clinical studies demonstrated the safety ofthe procedure.

The study protocol was that of an interventional case series in whichpatients with RP and a visual acuity of 20/800 or worse in one eye were studiedwithout a control group for comparison after approval from both Norton AudubonHospital, Louisville, Ky, and the University of Louisville human studies committee.The study was conducted under the Food and Drug Administration (FDA) investigationalnew drug number BB-IND 8354.

The patient who received the transplant gave informed consent afterextensive counseling regarding the realistic expectation of the procedure.In accordance with our selection criteria, this patient had a visual acuitymeasurement of 20/800 in the eye that was operated on for at least 1 yearwith a diagnosis of RP, was older than 21 years, was not pregnant, and waswilling to return for follow-up visits. The patient, a 64-year-old woman,was tested preoperatively on 3 separate occasions, 1 month apart, by EarlyTreatment Diabetic Retinopathy Study (ETDRS) so that she was accustomed tothe chart. The person measuring the ETDRS visual acuity was masked as to whicheye had the surgery. The patient underwent protocol refraction each time bythe masked observer. The ETDRS protocol for visual acuity testing was followedas previously described.⁸ The right eye, whichwas not operated on, was tested first.

Fetal tissue of 13 weeks' gestational age was obtained by informed consent.Donors were not compensated. After the donors had decided to terminate theirpregnancy, they were approached to donate tissue for research. The harvestingprocedure of fetal retina with RPE has previously been described.¹ A 1.5 × 3.1-mm piece of the fetal retina withits RPE was cut out and implanted subretinally under the fovea of the lefteye, using a custom-made implantation instrument with a flat plastic nozzletip at a 130° angle.^{1 ,6 - 7} Theloaded instrument was inserted under the retina through the retinotomy site,and the donor tissue was placed into the target area with the correct orientation,the RPE toward the choroid.

Donor tissue that was dissected away was used for the tissue typingof the donor at the histocompatibility lab at Jewish Hospital, Louisville.The donor DNA was extracted using a DNA tissue extraction kit (QIAGEN, Valencia,Calif) and typed for the HLA antigens HLA-A, HLA-B, and HLA-DR by polymerasechain reaction amplification using sequence specific primers (Pel Freeze,Brown Deer, Wis). Anti-HLA antigen antibodies in the recipient were detectedusing 2 techniques, both using sensitive flow cytometric procedures. Screeningfor antibodies was performed using a pool of normal T cells. The specificityof the antibody was determined using HLA antigen–coated beads (One Lambda,Canoga Park, Calif).

Complete assessments (ocular examination, fluorescein angiography, ETDRSprotocol for visual acuity testing, multifocal electroretinogram [mfERG],and multifocal visually evoked potential [mfVEP]) were performed preoperativelyand repeated postoperatively. To assess potentially corresponding physiologicchanges in the region of the transplant, photopic mfERGs and mfVEPs were recordedwith VERIS Science software (EDI Inc, San Mateo, Calif) using techniques andparameters previously described.^{7 ,9} Recordingswere performed 3 times before surgery and at 2 weeks and 1, 3, 6, and 12 monthspostoperatively.⁷

The patient was tested at 9 months postoperatively at an independenttesting site with the scanning laser ophthalmoscope (SLO) using a small, dimstimulus. The examiners at this location did not know which eye had receivedthe transplant. To compensate for the patient's eye movement after each measurement,the reference cross was fixed by manual tracking at clearly defined vascularlandmarks.¹⁰ The patient declined the opportunityfor preoperative testing, which makes this portion of the study difficultto interpret. However, the SLO testing postoperatively and the ETDRS testingcorrelated very well. The SLO testing was in addition to information obtainedfrom the other clinical tests and not in the experimental protocol.

The transplant could be observed easily after surgery by indirect ophthalmoscopybecause of its heavy pigmentation (Figure1A and B). During the follow-up examinations, the area of pigmentationeventually disappeared (between 3-6 months) (Figure 1C). The appearance of the fundus in the transplant arearemained unchanged between 6 and 12 months (Figure 1C and D). Fluorescein angiography showed no dye leakagein the area of the transplant at 6 to 12 months (Figure 2). There was no evidence of vitritis or vitreous cells.Although the clinical appearance of rejection in the retina is still somewhatunclear, no clinical evidence of tissue destruction, subretinal fibroses,or necrosis of the retina was seen in our patient. No systemic or intraocularimmunosuppression medications were used.

Tests of the medium surrounding the tissue before implantation for sterilityand endotoxin levels were well within normal limits (data not shown). TheHLA antigen types for donor and recipient are listed in Table 1. As expected, the donor and recipient were not matched.The donor and recipient shared only a single HLA-B antigen (B14) and no HLA-DRantigens, indicating this was an allogeneic graft.

No donor-specific antibodies were seen in the patient at 6 months. Anti–majorhistocompatibility complex class I antibodies specific for the HLA-A1 antigencarried on the graft were found before transplantation and did not changeat the time of the transplant. However, at approximately 3 months after transplantation,the titer of this class I antibody increased and continued to increase throughthe last sample collected 12 months after surgery. However, all other antibodies,including antibodies with specificities to antigens not expressed by the graft,increased concurrently. No anti–major histocompatibility complex classII antibody was detected at any time before or after the transplant.

The patient's vision in the eye that was operated on showed improvementboth subjectively and objectively, as assessed with ETDRS visual acuity testingat 7 months to 1 year. The patient declined the opportunity for preoperativeSLO testing but agreed to postoperative SLO testing when she noticed a markedsubjective improvement at 6 months. Objectively, her visual acuity improvedfrom 20/800 preoperatively to 20/400 at 6 months, 20/250 at 9 months, and20/160 at 12 months. No vision improvement occurred prior to 6 months. Incontrast, the ETDRS visual acuity of the eye that was not operated on, whichwas tested at every follow-up examination, did not change during the testingperiod from the 20/400 preoperative value. The postoperative ETDRS visualacuity at 9 months was confirmed by SLO testing at an independent clinicalcenter and measured 20/270 at a 40° field of view (Figure 3). The eye that was operated on contained 53 seeing and37 nonseeing areas (Figure 3). Thereappeared to be some fixation at the nasal edge of the transplant bed, andthere was a similar pattern of stability of fixation in both eyes. At 9 months,SLO testing of the eye that was not operated on measured the visual acuityat 20/369 (33 seeing and 37 nonseeing areas) (Figure 3), which was consistent with the visual acuity of 20/400in the same eye by ETDRS testing.

The overlay of the SLO fixation and the photograph of the transplantshow that the patient viewed the 20/270 letter (using 40° field of viewtesting) with a portion of the retina nasal to but at the edge of the transplant(Figure 4).The mfERG and mfVEP showedno clear signal preoperatively or postoperatively in any region of the retina.

Immediately after surgery, the patient required ventilation for pulmonaryedema following general anesthesia. This development did not appear to berelated to the instrument or fetal tissue retinal implant in her eye usedduring the retinal transplantation. Appropriate notification was made of thisadverse event to the FDA and institutional review board. The result of thisevent was that the patient had increased oxygen saturation levels for 5 dayspostoperatively in the range of 96.7% to 98.9% as measured by blood gasesand oximeter readings, which had been supplemented from a baseline of 92%saturation preoperatively.

The approach used in this patient with RP was to transplant an intactsheet of fetal neural retina and RPE. A specialized instrument and methodhave been developed to transplant sheets of fetal neural retina and RPE intothe subretinal space between the neurosensory retina and RPE. This approachhas significant advantages over other techniques because it maintains thecorrect orientation of the retinal sheets,^{6 ,11} andthe minimal trauma associated with the procedure reduces the possibility ofrosette formation or rupture of the Bruch membrane.

To date, no effective treatment has been developed for the recoveryof visual loss from RP, but the following 3 treatments are being investigated:(1) Oral vitamin A therapy has been demonstrated to be safe and effectivein slowing the rate of electroretinogram loss in RP but shows no effectivenessin the recovery of lost vision.¹² (2) Genetherapy and pharmacologic therapy are underway but are still under developmentand not in use in clinical trials at this time, although a clinical trialof gene therapy in Leber congenital amaurosis will probably be initiated in1 to 3 years.¹³ (3) Development and use ofa visual prosthesis is actively being pursued in many centers but the visualpotential of existing devices is not known.^{14 - 16}

In this patient, by 1 year postoperatively, no graft encapsulation,tissue destruction, or macular edema indicating rejection was seen clinicallyor with fluorescein angiography. However, one cannot exclude the presenceof more subtle graft rejection without histological data. Previous reportsof human transplantation have varied in the incidence of rejection dependingon whether the transplantation involved patches of RPE cells or dissociatedcells and whether the patients had exudative or nonexudative manifestationsof age-related macular degeneration.^{12 ,17 - 19} Theobserved pigment loss of the transplant might have been due to the death ofthe RPE cells (ie, graft failure) or to the fact that the pigment productionof the cells could not keep up with the growth of the cells. Based on clinicalexperience, depigmented RPE cells do not necessarily function normally. Pigmentloss has also been observed in cografts of rat fetal retina with RPE althoughthe donor RPE cells could still be identified by bromodeoxyuridine label.¹¹ However, it cannot be excluded that slow rejectionwas occurring. Perhaps there was a mild rejection response first manifestedas a loss of pigment. Nevertheless, fluorescein angiography showed no dyeleakage in the area of the transplant at 6 to 12 months.

The patient showed a general increase of major histocompatibility complexclass I antibodies at 3 months after transplantation, which indicates thatsome event stimulated the patient's immune system to produce antibodies. Whetherthe transplant or some other stimulus (eg, an illness) was responsible isunclear but it shows a nonspecific immunologic response as opposed to a graft-specificresponse. No new antibodies to graft-specific antigens developed as wouldbe expected in the presence of graft rejection. Also, the loss of pigmentin the graft occurred gradually across the 3- to 6-month period, and the beginningof the loss of pigment in the transplant was seen well before the titer increaseof the anti–class I antibody.

Taken together, our data indicate that changes in recipient antibodyproduction were not initiated by graft recognition and that graft-specificsensitization and thus rejection had not occurred in the observed time frame.

Because of the "immune privilege" of the subretinal space, retinal allograftsdo not elicit a classic immune response.^{20 - 22} Allogeneicsubretinal transplants of postnatal mouse retina (cell aggregates) containmicroglial cells expressing major histocompatibility complex class I and IIantigens at 35 days posttransplantation.²¹ Mostmicroglial cells are associated with blood vessels and migrate into the retinapostnatal in rats²³ and beginning at 16 weeks'gestation in humans.²⁴ Since the number ofmicroglial cells in fetal rat retina is much lower than in postnatal retina,²⁵ fetal retina, which still lacks inner retinal vessels,may be less immunogenic than postnatal rat retina. In an animal model usingallografts of Long-Evans or August Copenhagen Irish rat donors into Sprague-Dawleyor Royal College of Surgeons rat recipients, stable transplants were seenin rats 6 to 10 months after surgery,⁶ indicatingthat allogeneic retinal sheet transplants can be tolerated in the subretinalspace of rats with retinal degeneration. However, this does not necessarilyprove that no rejection occurred in the patient since results in animals cannotbe directly extrapolated to humans.

The subjective and objective visual acuity improvement appeared concurrentlyat 6 to 7 months after surgery. The patient's report of vision improvementwas corroborated by ETDRS visual acuity testing. The SLO testing indicateda similar result as the corresponding ETDRS protocol for visual acuity testing.However, since there was no preoperative testing, any improvement could notbe confirmed by SLO.

The SLO testing showed that fixation was unsteady and involved the nasaledge of the transplant as well as the retina nasal and immediately adjacentto the transplant. Viewing of the 20/270 letter was done with the retina atthe nasal edge of the transplant.

There are 2 mechanisms that may explain the visual improvement: a trophiceffect of the transplant on host cones^{26 - 27} orlocal synaptic connections between the transplant and host. Basic fibroblasticgrowth factor^{28 - 30} andvarious cytokines and neurotrophic factors³¹ havebeen shown to protect against photoreceptor degeneration due to continuouslight exposure or genetic defects.^{32 - 33} Thesegrowth factors likely act indirectly on photoreceptors via Mueller cells.³⁴ It has also been suggested that transplanted normalrod photoreceptor cells release soluble factor(s) to enhance cone survivalin primary rod photoreceptor dystrophies³⁵ withoutthe need for specific synapse formation.

An alternative or additional mechanism by which the transplant may haveimproved the vision in this patient is by local synaptic connectivity betweenthe transplant and host. However, this is uncertain in this case since thepatient fixated only on the edge of the graft. In Royal College of Surgeonsrats and transgenic rats with retinal degeneration, transplants of fetal retinalsheets can restore visual responses in an area of the superior colliculusthat topographically corresponds to the placement of the transplants in theretina.^{36 - 37} Preliminary studiessuggest synaptic connections between subretinal transplants and host retinaby a trans-synaptic virus tracing from the host brain to the transplant³⁸ (M.J.S., unpublished data, March 1999-June 2002).At the present time, however, there is no published experimental evidencethat transplanted fetal neurosensory retina can reestablish appropriate synapticconnections with the residual host neural network.

The failure of the mfERG to reveal improvement was not contradictoryto our conclusion of improved vision but rather indicative of an inabilityto extract any clear signal from the considerable recording noise (ie, a verylow signal-noise ratio). Because of the nature of the multifocal technique,any movement of the stimulus on the retina due to lack of good fixation willsmear the stimulation and response of functional areas of retina with adjacentnonfunctional areas. This effect will diminish any signal that may be present.The mfERG is additionally complicated by the issue of whether the waveformrecorded focally from the region of the transplant will resemble waveformscharacteristic of normal retinas.⁷ The waveformis likely to be affected by the types of connections made between the donorand host and precludes reliance on standardized templates in the analysis.Although no new tissue has been introduced to the visual cortex, waveformscomposing the normal mfVEP vary considerably across the visual field becauseof the convolutions of the primary visual cortex and individual variability.In patients, it is possible that transplant-induced changes in input and activityof the visual cortex after many years of their absence may result in neuralplasticity that produces waveforms uncharacteristic of normal mfVEPs. Theapproval of the FDA to perform this procedure in patients with better visualacuity (up to 20/400) and with less nystagmus will provide our future studieswith greater potential for showing improved function with mfERG and mfVEPtesting.

The effect of the patient's exposure to oxygen saturation levels at96.7% to 98.9% for 5 days postoperatively on the transplant is unknown.

Diseases that affect the RPE and photoreceptor cells of the retina (eg,RP, age-related macular degeneration, rod-cone dystrophy, and Stargardt disease)might conceivably benefit from this type of transplantation in the future.

An additional improvement has been seen in the patient presented inthis article since the time the manuscript was submitted. The patient wastested with follow-up ETDRS and SLO. The last SLO data presented in the articlewere at 9 months after surgery. At 1 year 3 months after transplantation,SLO testing showed a visual acuity of 20/260 in the eye that was operatedon and 20/330 in the eye that was not operated on with a 40° field ofview. At 2 years 3 months after surgery, SLO testing showed improved visualacuity in both eyes: 20/84 in the eye that was operated on and 20/139 in theeye that was not operated on. The fixation was similar over the transplantin both tests. At 2 years 2 months, ETDRS testing showed a visual acuity of20/200 in the eye that was operated on, whereas the visual acuity of the othereye remained unchanged at 20/400. Subjective improvement also occurred. At2 years 2 months after the surgery, the patient noted that she could definitelysee better with the eye that was operated on. The vision in that eye was lesscloudy than that of the other eye. She could also read the large-print Reader's Digest and print on the computer with the eyethat was operated on that she could not read with the other eye.

Correspondence: Norman D. Radtke, MD, 3 Audubon Plaza Dr, Suite 240,Louisville, KY 40217 (ret-vit.resource.ctr@prodigy.net).

Submitted for publication August 6, 2003; final revision received January28, 2004; accepted April 22, 2004.

This study was supported by The Murray Foundation Inc, New York, NY;the Vitreoretinal Research Foundation, Towson, Md; the Kentucky Lions EyeFoundation, Louisville; Research to Prevent Blindness, New York, NY; and ananonymous donor.

We thank Michael Lazar, project manager, Facilities Planning and Construction;Tatiana Forofonova, MD, PhD, and Marco Zarbin, MD, PhD, at The Universityof Medicine and Dentistry of New Jersey, New Jersey Medical School, Newark,for performing scanning laser ophthalmoscope testing, creating the overlayof the fundus photograph and scanning laser ophthalmoscope images, and interpretingthe scanning laser ophthalmoscope results.

Dr Radtke had full access to all the data in the study and takes responsibilityfor the integrity of the data and the accuracy of the data analysis.