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

RESULTS

Immediate Postoperative Results

Approximately 85% of the surgeries were completely successful (in a total of 38 eyes), with the construct injected clearly subretinally without complications. Otherwise, the most common complication observed was slight hemorrhage from a sclerotomy site in conjunction with surgery. However, the bleeding did not affect the final outcome of the surgery in any of the treated dogs. Further, in several cases the subretinal bleb did not form perfectly well, and most of the gene construct leaked out into the vitreous. The most serious immediate complication to the subretinal injection was a large partial retinal detachment that occurred in one case.

In nine of the treated dogs, postoperative intraocular inflammatory reactions (uveitis) were found to develop on the 2nd to 6th day in the rAAV.RPE65 treated eyes only (31,40,41). All were low grade except one, which was complicated with choroiretinitis and vitritis, refractory to treatment. The other eight cases of uveitis subsided after 4–12 wk of systemic and/or topical anti-inflammatory treatment.

Further investigation of the uveitis showed that these were most probably due to contaminating proteins in two of the four different batches of the rAAV.RPE65 gene construct used. Testing of the AAV showed that purity was approx 95% as estimated from both high-performance liquid chromatograms and silver-staining SDS polyacrylamide gels. Owing to problems with uveitis, batches used lately have been further tested using SDS-polyacrylamide gels and silver staining. Also filtering has been performed through a 0.45- m polyethersulfane (PES) membrane as well as screening for numerous viral and microbial contaminants by PCR. Because these rigorous measures have been taken, no further development of aberrant reactions following the gene transfer has occurred.

Early Postoperative Results

Clinical Findings and Visual Behavior

All dogs were systemically healthy postoperatively. Excellent functional improvement in visual behavior could be demonstrated as early as 4 wk following surgery in the previously blind affected dogs. The behavioral studies showed definite improvement of both day and night vision in dogs treated with subretinal injections of the rAAV.RPE65 gene construct. Vision appeared better in daylight than under dim light conditions. Preferential looking was observed from the side of the AAV.RPE65 treated eye only. Approximately 10 wk following the subretinal treatment with rAAV.RPE65, the nystagmus disappeared in both eyes in all affected dogs treated by unilateral subretinal gene transfer (31).

Early ERG Findings

Dark-adapted lowand high-intensity ERGs were mainly nonrecordable prior to treatment in the RPE65–/– dogs. Only a few of the affected dogs showed recordable scotopic low amplitude b-wave responses at the high intensity stimulus. In the light-adapted state, however, small amplitude responses, including single flash b-wave and 30 Hz flicker responses were obtained in many of the affected dogs. Fifty Hz flicker recordings were, however, not recorded in any of the affected dogs prior to the gene transfer treatment.

Fig. 2. (A) mfERG in progress with the dog under general anesthesia and mechanically ventilated (for maximum relaxation and no muscular movement). A SLO is used, equipped with an infrared laser for fundus visualization. (B) Fundus image with mfERG recordings from the left, nontreated eye and (C) similar recordings from the gene transfer treated eye. The mfERG stimulator is a prototype developed by Dr. Eric Sutter.

In all rAAV.RPE65 eyes treated with subretinal injections, the ERG amplitude responses improved 4–6 wk following surgery and statistically significant differences between AAV.RPE65 treated eyes and the contralateral control eyes were found. Also, ERG b-wave thresholds were close to normal in the treated dogs (42). ERG responses from lowand high-intensity scotopic light stimuli were obtained in treated dogs and photopic single flash, 30 and 50 Hz flicker responses were recorded in all gene transfer treated dogs performed successfully. The dark-adapted b-wave amplitudes had recovered to an average of 25% of normal, and the light adapted b-wave amplitudes to 20% of normal.

mf ERGs were performed in seven of the gene transfer treated RPE65–/– dogs 3–6 mo after surgery with continuous monitoring of the retinal position of the stimulus (38), to show the topographical restoration of visual function. A marked difference in response amplitudes in treated and untreated areas of eyes that had undergone gene transfer were found. An area of increased responses could be clearly correlated to the treatment area (Fig. 2). It was possible to obtain a focal response-intensity series in treated eyes, whereas the untreated eyes did not show such an increase in response amplitudes with increasing stimulus intensity at this time after surgery (3 mo) (Fig. 3). Although response amplitudes were greatest in the treated region, ERG responses were also detected when areas of the retina outside of the treated region was stimulated. This is consistent with a spreading of the treatment effect extending beyond the borders of the injection site.

Fig. 3. The topography of functional rescue was evaluated using bilateral mfERGs. An intensity series was performed in both eyes of unilaterally treated RPE65−/− dogs. This showed wave forms of increasing amplitudes in relation to increase in light intensity in the area of the gene transfer treatment in the right eye, depicted in the lower row of mf ERG responses.

Long-Term Postoperative Results

Visual Behavior

Six of the treated dogs were used for long-term follow-up. Using a maze test and observers blinded to the study up to 2 yr following gene transfer, objective visual testing was performed which demonstrated that the treated dogs had significantly improved vision over presurgical observations (34). Bright light vision was better than dim light vision, as evidenced by a significantly greater number of collisions with objects occurring under dim light conditions. Vision in control dogs (RPE65+/+) was not significantly different in dimand bright-light conditions. Further, although a significantly higher number of collisions were noted in the treated affected dogs than in normal control animals in dim light conditions, in bright light conditions the affected dogs that had undergone gene therapy performed as well as normal control dogs. Although vision in treated dogs was not completely normal, especially in dim light, these results represent a potentially dramatic improvement in the general quality of life as a result of the gene therapy.

ERG

The maximum improvement of rod ERG responses was found at 3 mo after gene transfer. The dark-adapted b-wave maximum amplitudes recovered to an average of 28% of normal, and the light-adapted b-wave maximum amplitudes to 47% of normal (31,32). After 3 mo, there was a slight reduction in rod responses over time, and at 18 mo postsurgery, scotopic high-intensity b-wave amplitudes, photopic single-flash and 30 Hz flicker b-wave amplitudes were still significantly increased above baseline values. For the cone system, there was a slow, long-term improvement, which was either sustained or continued to increase up to 18 mo following the gene transfer treatment. By 21 to 24 mo following treatment, rod responses and maximum b-wave amplitude values in the treated eye had declined further, and continued to decrease in amplitudes at 24 and 33

Fig. 4. The actual ERG recordings from a normal control dog, far left, and an affected gene transfer treated littermate before surgery (pre) and at 1, 9, 15, 21, and 27 mo postoperatively. The affected dog was injected with the gene construct (rAAV.RPE65) subretinally into the right eye and, in a similar way, with rAAV.GFP into the left eye. The latter construct was used in the right eye (only) of the control dog. Upper recording in each set of two traces are ERG responses from the right eye and lower recording from the left eye. The simultaneous bilateral full-field ERG responses from each animal were obtained using scotopic low-light intensity stimuli (–2 log cd s/m2); 1st row of responses, and scotopic high-light stimuli levels (4 cd s/m2); 2nd row. After 10 min of light adaptation photopic single-flash stimulation was used (5 Hz) at 1 cd s/m2; 3rd row, then flicker stimulation at 30 and 50 Hz, using 1cd s/m2 of light stimulation; 4th and 5th rows, respectively.

mo after surgery. Cone responses, indicated by photopic single-flash b-wave responses were still significantly elevated over preoperative values up to 30 mo following surgery. At 33 mo following surgery, however, no significant differences were found for either rod or cone response amplitudes in comparison to preoperative ERG recordings (43) (Fig. 4).

An Unexpected But Positive Finding

An unexpected finding was encountered at long-term follow-up in five of the dogs used for long-term studies: there was a surprising appearance of low-amplitude ERG responses (at high-intensity scotopic and photopic recordings) also in the fellow eyes starting approx 6–9 mo after surgery. This contralateral eye effect was sustained (for 30 Hz flicker responses) up to 27–30 mo after treatment. Although these positive ERG responses were of low amplitude in the fellow eyes, there was a definite qualitative improvement of these responses compared to preoperative responses, most of which were nonrecordable.

Through careful control studies of the contralateral eye effect we can conclude that the positive ERG responses seen on the contralateral (control) eye at long-term followup was real and not an artifact, and appears as a result of the gene transfer treatment. These results could indicate transmission of RPE65 protein, of the gene transfer construct, or most likely, of 11-cis-retinal to the fellow eye, and suggest that cells outside of the treated area may also benefit from the rAAV.RPE65 gene therapy. This is of particular importance in adapting this therapeutic approach to humans because the most

important region of the retina in which to restore function is the macula. However, it would be detrimental to inject the construct in the macular area owing to the risk of causing permanent damage from the temporary retinal detachment. If the treatment effect can be detected in the untreated eye, surely it would extend beyond the injection site in the treated eye. Thus, it is likely that a subretinal injection using a gene therapy construct peripheral to the macula would also restore macular function.

Wide Time Window for Surgery

Subretinal gene transfer treatments were given to two older dogs, the oldest being 4 yr old at the time of surgery, without complications. Both dogs showed a markedly improved visual behavior 4–6 wk postoperatively and increased ERG sensitivity and amplitudes as previously described (42). There was no obvious difference in visual behavior upon follow-up of these older dogs (now 39 and 7 mo after surgery, respectively), compared to younger treated animals. It appears that components of the phototransduction cascade and the retinal circuitry remain functional, despite the absence of normal photoreceptor activity for several years in untreated dogs. As has been shown for Rpe65–/– mice, injections of 11-cis-retinal result in regeneration of rhodopsin and improved photoreceptor function—irrespective of age (44). Thus, there is a wide time window for surgery in conjunction with the RPE65 null mutation, a fact that has positive implications, also for human patients.

Effect of Subretinal Gene Therapy on In Vivo Retinal Morphology

In order to determine the magnitude and distribution of GFP transduced retinal cells (Fig. 5) retinal fluorescence was examined in vivo and detailed fundus images obtained using the SLO, 2 yr following the subretinal injection. Transduction was still obvious in the left eye, as observed by the GFP fluorescence in the area of the subretinal (control) injection. Further, retinal morphology following gene transfer studies were performed in treated eyes using the SLO and FL and ICG angiography, 2 to 2.5 yr after treatment. Surprisingly, good SLO images were obtained with no major alterations observed in the injection area in five long-term studied dogs. However, when using FL and ICG angiography, there were marked changes. Specifically at the injection site, in a region smaller than the original bleb area, there was a distinct part with successively increasing hyperfluorescence, possibly indicating leakage from retinal vessels and/or atrophic focal changes in the RPE (Fig. 5). These observations are potentially of major significance for application of this treatment to human subjects. Damage to the retina at the injection site would indicate that injecting the construct too near the macula should be avoided. The fact that abnormalities were restricted to the injection site indicates that there is no geographic spreading of this negative effect, however.

Light and Electron Microscopy of Treated Dogs

So far six RPE65–/– dogs given subretinal injections of the AAV-RPE65 gene therapy vector have been analyzed for the effects of treatment on retinal morphology. The first dog was euthanized 3 mo after the gene therapy treatment. Modest recovery of visual sensitivity was observed in this dog in the treated eye, as measured with ERG (31). The dog had severe uveitis in the AAV-RPE65 treated eye that did not respond to

Fig. 5. SLO images of a gene transfer treated dog 2 yr after rAAV.RPE65 into the right eye and rAAV.GFP into the left eye, both injections given subretinally. (A) Transduction is still obvious in the left eye, as observed by AAV.GFP fluorescence in an area close to the optic disc, although significantly less than at 3 mo following surgery (for 3-mo follow-up, see ref. 31). (B) In the subretinal rAAVRPE65 bleb area (inferior-nasal to the disc) in the right eye, 2 yr after treatment, there are only minor changes and severe scarring was not observed. (C) Fluorescein angiography, however, in this gene transfer treated eye shows a late hyperfluorescence in the neuroretinal bleb area, indicating distinct morphologic changes specifically at the injection site.

intensive topical and systemic anti-inflammatory medication. The clinically observed uveitis was accompanied by lymphocytic infiltration along the inner retinal surface. In the AAV-RPE65-treated eye, RPE65 immunolabeling indicated that RPE65 protein was being produced by the RPE only in the treated region of the eye (31,33). In the fellow eye, GFP expression was pronounced in the RPE throughout the region where the subretinal bleb had been made. Electron microscopic analysis demonstrated that in the fellow eye the RPE contained numerous large lipid droplets in both injected and un-injected regions of the retina. In the eye treated with the AAV-RPE65 vector, the lipid droplets had almost completely disappeared from the RPE in the treated region. However, outside of the treated region, the RPE still contained large numbers of inclusions (31). Despite reversal of the RPE lipid droplet accumulation in the treated region, there was no apparent recovery of photoreceptor OS morphology 3 mo after surgery. Thus, the observed functional recovery, measured by ERG, must have been mediated by the remaining OS fragments in the treated region or by photoreceptor cells outside of the treated region.

A littermate to the first dog was euthanized 6 mo after gene transfer surgery and a third dog was euthanized 5 mo after the treatment. None of the eyes of these dogs had been affected by uveitis. The eyes of the latter dog were used primarily for immunocytochemistry studies (performed this time at the Wallenberg Retina Center at the University of Lund, Sweden). At 5 mo postsurgery, a significant amount of RPE65 immunolabeling was observed in the RPE of the treated eye, but none in the contralateral eye (33). There was also a marked reduction of lipoid inclusions in the RPE in the treated eye, and further, an obvious difference between the structure of cone OS in the

Fig. 6. Ultrastructural studies showed large lipoid inclusions (L) in the RPE of an untreated RPE65–/– dog (left image), littermate of a treated dog (right image) that had undergone gene transfer 10 mo earlier. In the latter dog there was a lack of lipoid inclusions in the area of the gene construct injection. Note also the normal appearing photoreceptor outer segments in this dog in comparison to those of the untreated littermate. Courtesy of Dr. Martin Natz.

treated eye compared to those of the untreated eye. In the gene transfer treated eye, the cones seemed to be aligned more orderly and appeared more robust than in the contralateral, untouched eye.

Morphological analysis was performed also on the eyes of two RPE65–/– littermates that were euthanized 10 mo after administration of the AAV vectors into one of the dogs. The untreated littermate was used as a control. The dog that had undergone gene transfer had shown a low-grade, transient uveitis in the AAV-RPE65 treated eye. Longterm follow-up ERG studies showed restoration of photoreceptor function, not only in the treated eye, but also slight recovery of photopic responses in the contralateral untreated eye, starting 6 mo postsurgery. Ultrastructural studies were undertaken to determine whether there were morphological changes that correlated with the functional recovery of the eyes. In the rAAV.RPE65 treated eye, almost no RPE lipoid inclusions characteristic of the RPE65 null mutation were observed within the treated region (45). In the opposite, untreated eye, the numbers and sizes of lipid droplets were diminished relative to those present in the eyes of the untreated littermate. Rod and cone OS morphology appeared orderly and elongated in the treated eye compared to that of the untreated dog and the fellow eye (Fig. 6). These preliminary results for the gene therapy treated eye establish a basis for the functional recovery of scotopic and photopic ERG responses obtained upon clinical follow-up in the gene transfer treated eye.

These results were further verified when another dog used for long-term follow-up was euthanized 2.5 yr following the gene transfer (46). This dog had exceptionally highamplitude ERG responses in the gene transfer treated eye, as well as increased mainly photopic ERG responses in the fellow eye. Following light and electron microscopic

studies of the retina, several exciting results were obtained: the photoreceptor outer and inner segments were orderly aligned in areas of the retina peripheral to the immediate injection site. The latter area was, however, morphologically disrupted. Outside of the immediate injection area the outer segments were normally elongated toward the RPE, and contained stacks of normally oriented lamellar discs. Lipoid inclusions were abolished in the former bleb area although still prevalent in more peripheral regions. Further studies are in progress with morphometric analysis of tissue from both eyes in order to evaluate the spatial distribution of treatment effects.

DISCUSSION AND CONCLUSION

Gene therapy in dogs affected with a hereditary retinal dystrophy caused by the RPE65 null mutation resulted in a remarkable improvement in visually mediated behavior and in retinal function as assessed by ERG. Rescue of visual function was observed in all dogs in which the vector was successfully delivered subretinally. Immunocytochemical analysis detected RPE65 expression only in the area of the RPE where the gene vector was applied and it was in this specific area that the lipoid inclusions were markedly diminished after treatment. The immediate injection site showed several alterations in retinal morphology as visualized at long-term follow-up by FL and ICG angiography. On the other hand, photoreceptor OS morphology appeared normalized by the gene transfer both within and outside of the treated region. The only adverse effects in the gene therapy treatment observed were transient inflammatory responses in the eyes of some of the injected animals as a result of impurities in the gene therapy vector preparations.

The therapeutic expression of RPE65 from an rAAV vector injected subretinally in RPE65–/– dogs was effectively achieved up to approx 30 mo following gene therapy. The effect of treatment was successively reduced to levels where ERG amplitudes were insignificantly increased in comparison to preoperatively. The reason for this diminished functional effect is not clear, but may be because of several factors such as promoter shut-off or transgene silencing (47–50). Another reason for the reduced effect could be the loss of transgene expressing cells since long-term follow-up studies have clearly shown that there is a progression of clinical disease in the 3 yr following the initial gene transfer treatments (Fig. 1).

The contralateral eye effect shows up as a low-grade qualitative improvement of the ERG responses observed additionally in the fellow eyes of gene transfer treated dogs. The phenomenon appears to be most prominent on cone function and the effect is transient. The precise mechanism for this effect in the presently treated group of RPE65–/– dogs has not been elucidated, but there may be several possible explanations. It has been shown that unilateral cell injury causes multiple cellular responses in the contralateral eye (51). Further, unilateral pseudorabies virus injections with GFP into the vitreous of one eye in hamsters infected the intergeniculate leaflet of the thalamus. It was later found that the retinal ganglion cells in the contralateral eye also expressed GFP, becoming infected after transsynaptic uptake and retrograde transport through infected retinorecipient neurons (52). Our findings and those of others, thus clearly indicate that the contralateral or fellow eye, even if untreated, should not be used as a “control” eye. Gene transfer in the presently described unilaterally treated dogs resulted in the production of 11-cis-retinal, which long-term could be disseminated to the contralateral eye in small amounts, the mechanism of which is still obscure.

AAV-mediated gene therapy is an effective means of rescuing visual function in inherited diseases that result in retinal dysfunction. Modification of the gene construct may be necessary to achieve more long-term results of the treatment. However, the subretinal approach of treating outer retinal disease appears safe as long as the vector preparations are of high purity and are not given directly into the macular region.

ACKNOWLEDGMENTS

We would like to acknowledge the following coworkers, all of which have been involved in some parts of this project: Ragnheidur Bragadottir, MD, PhD, Norway; Anitha Bruun, PhD, Sweden; Lynnette Caro, DVM, USA; Marnie Ford, DVM, PhD, Canada; Martin Katz, PhD, USA; Helmut M. Mayser, MD, Germany; Piroska E. Rakoczy, PhD, Australia; T. Michael Redmond, PhD, USA; Ernst-Otto Ropstad, DVM, Norway; Vaegan, PhD, Australia.

For superb technical assistance, we would like to thank Jenny Garland, Ginny Dodam, Leilani Castaner, Deborah Becker, and Howard Wilson. We are grateful to the University of North Carolina Gene Therapy Vector Core facility of Dr. R. Jude Samulski, PhD, for providing the SSV9 plasmid.

This work was supported by the Foundation Fighting Blindness, Research to Prevent Blindness, Inc., and the University of Missouri Research Board.

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