Long-Term Protection of Retinal Structure but Not Function Using ...

doi:10.1006/mthe.2001.0473, available online at http://www.idealibrary.com on IDEAL

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Long-Term Protection of Retinal Structure but Not Function Using RAAV.CNTF in Animal Models of Retinitis Pigmentosa Fong-Qi Liang,1 Tomas S. Aleman,1 Nadine S. Dejneka,1 Lorita Dudus,2 Krishna J. Fisher,2 Albert M. Maguire,1 Samuel G. Jacobson,1 and Jean Bennett1,* 1

F. M. Kirby Center for Molecular Ophthalmology, Department of Ophthalmology, Scheie Eye Institute, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA 2 Department of Pathology and Laboratory Medicine, Tulane University Medical Center, New Orleans, Louisiana 70112, USA *To whom correspondence and reprint requests should be addressed. Fax: (215) 573-7155. E-mail: [email protected].

The present study aimed to determine whether intravitreal administration of an adeno-associated virus (AAV) carrying ciliary neurotrophic factor (CNTF) can achieve long-term morphological and physiological rescue of photoreceptors in animal models of retinitis pigmentosa, and whether injection of this virus after degeneration begins is effective in protecting the remaining photoreceptors. We injected rAAV.CNTF.GFP intravitreally in early postnatal Prph2Rd2/Rd2 (formerly rds/rds) mice and in adult P23H and S334ter rhodopsin transgenic rats. Contralateral eyes received an intravitreal injection of rAAV.GFP or a sham injection. We evaluated the eyes at 6 months (rats) and 8.5 to 9 months (mice) postinfection and looked for histological and electoretinographic (ERG) evidence of photoreceptor rescue and CNTF–GFP expression. Intravitreal administration of rAAV resulted in efficient transduction of retinal ganglion cells in the Prph2Rd2/Rd2 retina, and ganglion, Muller, and horizontal/amacrine cells in the mutant rat retinas. Transgene expression localized to the retinal region closest to the injection site. We observed prominent morphological protection of photoreceptors in the eyes of all animals receiving rAAV.CNTF.GFP. We found the greatest protection in regions most distant from the CNTF–GFP-expressing cells. The Prph2Rd2/Rd2 ERGs did not exhibit interocular differences. Eyes of the rat models administered rAAV.CNTF.GFP had lower ERG amplitudes than those receiving rAAV.GFP. The discordance of functional and structural results, especially in the rat models, points to the need for a greater understanding of the mechanism of action of CNTF before human application can be considered. Key Words: CNTF, AAV, photoreceptor, trophic factors, retinitis pigmentosa

INTRODUCTION Retinitis pigmentosa (RP) is a group of inherited retinal degenerative diseases that lead to progressive reduction in visual field extent and impairment of visual acuity. The visual deficits in RP are caused by photoreceptor degeneration, which is triggered by mutations in a variety of different genes [1]. Although the mechanisms by which these genetic insults lead to photoreceptor death are still not clear, the final common pathway is apoptosis [2,3]. Therefore, modulation of photoreceptor apoptosis may offer an effective therapeutic approach to RP. A number of trophic factors, including ciliary neurotrophic factor (CNTF), promote photoreceptor survival in animal models of retinal degeneration [4–7]. For example, intravitreal injections of CNTF protein led to transient pro-

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tection of photoreceptors in Pde6brd1/rd1, nr/nr, and rhodopsin Q344ter mutant mice, but not in other animal models [4]. Lack of protection in other models may be due to the short biological half-life of CNTF and species/strain differences. Thus a delivery system which could increase its availability over longer time periods may be more effective. CNTF slowed photoreceptor degeneration in Prph2Rd2/Rd2 mice following delivery with a recombinant adenovirus [8]. Recent reports also indicated that recombinant adeno-associated virus (AAV) or CNTF-producing cells delay photoreceptor degeneration in P23H and S334ter rhodopsin transgenic rats and RCD1 dogs, respectively [9–11]. These findings suggest that a continual supply of CNTF can prolong photoreceptor survival in other animal models.

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FIG. 1. Light photomicrographs of eyecups (A, B) from a representative 8.5-month-old Prph2Rd2/Rd2 mouse that received an intravitreal injection of rAAV.CNTF.GFP in one eye (A) and rAAV.GFP in the contralateral eye (B) at postnatal day 5. Note the difference in ONL thickness between the temporal (t) and nasal (n) retina of both eyes (top). (C–F) Higher magnification views of the central areas from each hemisphere (C, D, nasal and temporal central area of (A), respectively; E, F, nasal and temporal central area of (B), respectively). Significant preservation of photoreceptors was seen in the nasal retina (C) compared with the temporal retina (D) in the rAAV.CNTF.GFP injected eye (A). Photoreceptors degenerated in both hemispheres (E, F) of the rAAV.GFP injected eye (B), with approximately one row of nuclei remaining. onl, outer nuclear layer; inl, inner nuclear layer; gcl, ganglion cell layer; rpe, retinal pigment epithelium; on, optic nerve. Bar, 20 m.

We previously generated an AAV expressing a secretable form of CNTF in tandem with the marker green fluorescent protein (GFP). Subretinal administration of this virus (rAAV.CNTF.GFP) led to efficient transduction and protection of photoreceptor cells (for 3 months) in rho–/– and Prph2Rd2/Rd2 mice as well as in P23H mutant rhodopsin transgenic rats [12,13]. Rescue was limited to a local area of the retina and was likely due to the subretinal approach, which allows delivery of a very small volume of viral solution to an artificial space limited by cellular boundaries. To achieve a wider retinal extent of rescue, delivery of larger volumes of transduction solution or multiple subretinal injections may be required. Alternatively, intravitreal injection may be a more efficient route for greater therapeutic effects. This approach allows delivery of a larger volume of solution, which can potentially expose the entire retina. AAV delivered intravitreally can target retinal ganglion cells [14], and thus it may be feasible to harness retinal ganglion cells to produce CNTF for therapeutic purposes. Diffusion of secreted CNTF across the retina could potentially exert a protective effect on photoreceptors because recent studies suggest that CNTF-induced photoreceptor protection occurs indirectly

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through activation of retinal ganglion, Muller, and perhaps other non-photoreceptor cells [15–17]. Accordingly, our study aimed to determine whether intravitreal administration of AAV.CNTF.GFP can achieve broad and long-term morphological and functional rescue of photoreceptors in animal models of RP, and whether this virus can protect the remaining photoreceptors following the start of retinal degeneration. This approach may ultimately be useful as a generic treatment for human disease, preserving photoreceptors regardless of genetic defects, and may substantially advance the treatment of RP.

RESULTS Histological Protection of Photoreceptors Throughout the experiment the animals showed no obvious signs of ocular inflammation. We observed photoreceptor protection by morphological criteria in all three animal models. The Prph2Rd2/Rd2 mouse (also known as retinal degeneration slow, rds/rds) carries a spontaneous null mutation of the gene Prph2. Consequently, these mice fail to develop photoreceptor outer segments. Photoreceptors

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FIG. 2. Comparison of the ONL thickness (m) and number of rows of nuclei in the ONL in the temporal and nasal halves of retina as well as the whole retina in 8.5 to 9months-old Prph2Rd2/Rd2 (rds/rds) mice (n = 13) that received intravitreal injections of AAV.CNTF.GFP (OD) and AAV.GFP/sham (OS) at postnatal days 3–5. Numbers represent mean ± standard deviation (SD). *P < 0.01 compared with the contralateral eye. #P < 0.01 compared with the nasal retina of the contralateral eye, but P < 0.05 when compared with the temporal retina of same eye. **P < 0.05 compared with the temporal retina of the contralateral eye. OD, right eye; OS, left eye.

degenerate slowly and totally disappear by the age of 8–11 months [18]. Histological examination showed obvious preservation of photoreceptor nuclear layers in the rAAV.CNTF.GFP injected eyes (Fig. 1A) in comparison with rAAV.GFP injected eyes (Fig. 1B). We did not observe outer segments in either rAAV.CNTF.GFP or rAAV.GFP injected eyes. Morphometric analyses revealed significant differences in the thickness of the outer nuclear layer (ONL) and the number of rows of nuclei in the ONL between the rAAV.CNTF.GFP-injected and contralateral control eyes (P < 0.01; Fig. 2). There were large standard deviations of ONL thickness in the eyes receiving rAAV.CNTF.GFP injections. This was mainly due to the large differences in ONL thickness between the temporal and nasal retina as well as variability among eyes. The nasal half of the retina in the rAAV.CNTF.GFP-injected eye displayed a significantly thicker ONL and greater number of rows of photoreceptor nuclei compared with the temporal hemisphere of the same eye (P < 0.05) and the contralateral eye (P < 0.01, Fig. 2). Typically, the nasal retina of the rAAV.CNTF.GFP-injected eyes possessed four to five rows of photoreceptor nuclei (Fig. 1C), as opposed to two to three rows in the temporal retina of the same eye (Fig. 1D) and approximately one row in the entire retina of the contralateral eye (Figs. 1E and 1F). The thickness of the ONL and the number of rows of photoreceptor nuclei in the temporal retina of AAV.CNTF.GFP-injected eyes were significantly decreased compared with those of the nasal retina of the same eyes; however, they possessed significantly more rows than the contralateral eyes (P < 0.05; Fig. 2). We did not detect significant differences between the protected regions of the temporal and nasal hemispheres of the retina in the contralateral eyes. Both had significantly fewer photoreceptors than the rAAV.CNTF.GFPinjected eyes. Heterozygous P23H and S334ter transgenic rats carry a mutated opsin gene in addition to the endogenous native opsin gene [19]. Photoreceptors in these animals undergo slow degeneration, with 1–2 rows of nuclei remaining at

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the age of 3 months [20,21]. Rhodopsin transgenic rats used here are homozygous, so photoreceptors may degenerate faster. Similar to the Prph2Rd2/Rd2 mice, histological examination revealed significant protection of photoreceptors in the rAAV.CNTF.GFP injected eyes of P23H (Fig. 3A) and S334ter (Fig. 5A) rats, but not in the contralateral eyes, which received rAAV.GFP or sham injections (Figs. 3B and 5B). However, we observed substantially greater hemispheric differences in photoreceptor protection in these transgenic rats compared with those in Prph2Rd2/Rd2 mice. In the rAAV.CNTF.GFP injected eyes, the nasal retina showed a significantly thicker ONL and greater number of rows of photoreceptor nuclei as compared with those in the temporal halves of the retinas (P < 0.01; Figs. 4 and 6). Typically, the nasal retina possessed three to four rows in the ONL (Figs. 3C and 5C) as opposed to one row or less in the temporal retina of the rAAV.CNTF.GFP-injected eyes (Figs. 2D and 3D). In the contralateral control eyes, there was one row or less remaining in the ONL across the entire retina (Figs. 2E, 2F, 5E, and 5F), which was similar to that in the temporal retina of the rAAV.CNTF.GFP injected eyes (P = 0.82). rAAV-Mediated Gene Transfer in the Retina To determine what types of retinal cells were transduced by rAAV after intravitreal administration, we examined GFP fluorescence histologically. Fluorescent microscopy showed that GFP was strongly expressed in retinal ganglion cells in both rAAV.CNTF.GFP- and rAAV.GFPinjected eyes (Figs. 7A and 7B). Cells in the temporal retina corresponding to the site of injection expressed GFP. Most GFP fluorescent cells localized to a region comprising approximately one-half of the temporal hemisphere. We also observed GFP fluorescence in the nerve fiber layer of the temporal retina and the temporal side of the optic nerve. Additionally, some RPE and photoreceptor cells showed GFP expression in the injection site. This was likely due to leakage of viral solution into the subretinal space during needle entry and withdrawal, and subsequent infection of RPE and photoreceptors. Occasionally, we

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FIG. 3. Light photomicrographs of eyecups (A, B) from a representative 6-month-old P23H transgenic rat that received an intravitreal injection of rAAV.CNTF.GFP in one eye (A) and rAAV.GFP in the contralateral eye (B) at postnatal day 28. Note the difference in ONL thickness between the temporal (t) and nasal (n) retina of (A). (C–F) Higher magnification views of the central areas from each hemisphere (C, D, nasal and temporal central area of (A), respectively; E, F, nasal and temporal central area of (B), respectively). Significant preservation of photoreceptors was seen in the nasal retina (C). Photoreceptors in the temporal retina (D) degenerated similarly to those in the nasal and temporal hemisphere (E, F), with approximately 1 row of nuclei remaining. onl, Outer nuclear layer; inl, inner nuclear layer; gcl, ganglion cell layer; rpe, retinal pigment epithelium; on, optic nerve. Bar, 20 m.

identified GFP-expressing cells in the ciliary body. We did not observe GFP in the nasal retina or other parts of the eye which had been exposed to either rAAV.CNTF.GFP or rAAV.GFP. Intravitreal injection of rAAV.GFP in adult Prph2Rd2/Rd2 mice also resulted in GFP expression in Muller cells (data not shown). We saw GFP fluorescence predominantly in the temporal retina in both rAAV.CNTF.GFP- and rAAV.GFP-injected eyes of P23H and S334ter rats (Figs. 7C and 7D), which was similar to the transduction seen in the Prph2Rd2/Rd2 animals.

However, GFP-expressing cells localized to a larger area, from one-half to the entire temporal half of the retina. In addition to ganglion cell transduction, we observed strong GFP expression in the Muller-like cells (Figs. 7C and 7D). Some horizontal/amacrine cells, identified by their location and horizontal processes, also showed GFP expression (Fig. 7C). GFP fluorescence was apparent in the nerve fiber layer of the temporal retina and the temporal side of the optic nerve (Fig. 8). Retinal ganglion cells rarely expressed GFP in the nasal retina. GFP was not observed in other parts of the eye.

FIG. 4. Comparison of the ONL thickness (m) and number of rows of nuclei in the ONL in the temporal and nasal halves of retina as well as the whole retina in 6month-old P23H rhodopsin transgenic rats (n = 9), which received intravitreal injections of AAV.CNTF.GFP (OD) and AAV.GFP/sham (OS) at postnatal days 28–35. Numbers represent mean ± SD. *P < 0.05 compared with the contralateral eye. #P < 0.01 compared with the temporal retina of same eye and the nasal retina of contralateral eye.

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FIG. 5. Light photomicrographs of eyecups (A, B) from a representative 6-month-old S334ter transgenic rat that received an intravitreal injection of rAAV.CNTF.GFP in one eye (A) and rAAV.GFP in the contralateral eye (B) at postnatal day 28. Note the difference in ONL thickness between the temporal (t) and nasal (n) retina of (A) . (C–F) Higher magnification views of the central areas from each hemisphere (C, D, nasal and temporal central area of A, respectively; E, F, nasal and temporal central area of B, respectively). Significant preservation of photoreceptors was seen in the nasal retina (C). Photoreceptors in the temporal retina (D) degenerated in a similar fashion to those in the nasal and temporal hemisphere (E, F), with approximately 1 row of nuclei remaining. onl, outer nuclear layer; inl, inner nuclear layer; gcl, ganglion cell layer; rpe, retinal pigment epithelium; on, optic nerve. Bar, 20 m.

To confirm Muller cell transduction by rAAV, we performed immunostaining for glial fibrillary acidic protein (GFAP), a marker for glial cells, in the retinal sections of P23H and S334ter eyes that received an rAAV injection. We observed prominent GFAP immunoreactivity in the nerve fiber layer and Muller cell end feet (Fig. 9A). Muller cell bodies and processes co-expressed GFAP and GFP (Figs. 9A and 9B). To verify that the GFP-expressing Muller-like cells were not bipolar cells, we immunostained with a monoclonal antibody directed against protein kinase C (PKC), a marker for bipolar cells. We did not observe colocalization of GFP and PKC in GFP-expressing Muller-like

cells, but we detected PKC immunoreactivity in the inner nuclear layer and ganglion cell layer in the rAAV injected eyes (data not shown). CNTF Expression To verify whether photoreceptor protection was due to CNTF production in the rAAV.CNTF.GFP injected eyes, we carried out immunohistochemistry using a commercially available antibody (anti-rat CNTF, Promega). Retinal regions showing GFP expression (that is, temporal retina) in the rAAV.CNTF.GFP injected eyes were CNTF positive. Strong CNTF immunoreactivity localized to the nerve

FIG. 6. Comparison of the ONL thickness (m) and number of rows of nuclei in the ONL in the temporal and nasal halves of the retina as well as the whole retina in 6-month S334ter rhodopsin transgenic rats (n = 7), which received intravitreal injections of AAV.CNTF.GFP (OD) and AAV.GFP/sham (OS) at postnatal days 28–35. Numbers represent mean ± SD. *P < 0.05 compared with the contralateral eye. #P < 0.01 compared with the temporal retina of same eye and the nasal retina of contralateral eye.

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FIG. 7. Presence of GFP in retinal cells infected with rAAV.CNTF.GFP (A, C) and rAAV.GFP (B, D) in an 8.5-month Prph2Rd2/Rd2 mouse and a 6-monthold P23H rat. Sections were counterstained with propidium iodine and viewed under a dual filter (FITC/rhodamine). Pictures were taken from the temporal central region of each eye. In the Prph2Rd2/Rd2 eye (A, B), GFP fluorescence was seen only in the ganglion cells, whereas in the P23H rat (C, D), Muller cells (arrows) and amacrine cells (C, identified according to their retinal location) were also transduced.

fiber layer and ganglion cell layer, and colocalized with GFP (Figs. 10A and 10B). GFP-expressing Muller cells (Fig. 10C) also displayed prominent CNTF immunoreactivity (Fig. 10D). In contrast, CNTF immunoreactivity in the nasal retina of the rAAV.CNTF.GFP injected eyes was minimal, similar to that of the entire retina of rAAV.GFP injected eyes. Retinal Function To determine if there was functional as well as structural protection, we determined interocular differences in electroretinogram (ERG) amplitude between rAAV.CNTF.GFPinjected and rAAV.GFP-injected eyes in animals representing each of the three groups (Fig. 11). Of six Prph2Rd2/Rd2 mice, three did not have detectable ERGs in both eyes. The other three animals possessed small detectable amplitudes in each eye. We did not find significant differences

in b-wave amplitudes between rAAV.CNTF.GFP-injected eyes (mean = 5 V, SEM = 2 V) and rAAV.GFP-injected controls (7 ± 2 V). The P23H rhodopsin transgenic rats had statistically significant interocular differences in ERGs; there were smaller b-wave amplitudes in rAAV.CNTF.GFP eyes (4 ± 1 V) than in the rAAV.GFP eyes (12 ± 3 V). A similar data trend occurred in the S334ter rhodopsin transgenic rats. We observed significantly smaller ERG amplitudes in the rAAV.CNTF.GFP injected eyes (8 ± 3 V) versus rAAV.GFP injected eyes (24 ± 2 V). Flicker ERGs (data not shown) generally followed the results of the darkadapted responses. Prph2Rd2/Rd2 animals did not possess measurable flicker waveforms, whereas P23H rats showed statistically significant interocular differences in ERGs (P = 0.01), with smaller amplitudes in rAAV.CNTF.GFP eyes (0.3 ± 0.2 V) than in the rAAV.GFP eyes (2.0 ± 0.5 V). S334ter rats also possessed significant interocular differ-

FIG. 8. Presence of GFP in the nerve fiber layer (nfl) in the temporal (T) retina and optic nerve (on) in the rAAV.CNTF.GFP injected eye of a 6-month-old P23H rat. Note lack of GFP fluorescence in the nasal (N) retina and the difference in the ONL thickness across the optic nerve. Autofluorescence appears in the RPE. onl, outer nuclear layer; inl, inner nuclear layer; gcl, ganglion cell layer; rpe, retinal pigment epithelium; on, optic nerve.

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DISCUSSION

FIG. 9. Co-localization of GFAP (A) and GFP (B) in a Muller cell (arrows). A and B represent the temporal region of retina in the rAAV.CNTF.GFP-injected eye of a 6-month-old P23H rat viewed under rhodamine (A) and FITC (B) filters.

ences in flicker ERGs (P = 0.002), with amplitudes in the rAAV.CNTF.GFP-injected eyes (1.1 ± 0.4 V) being smaller than those in rAAV.GFP-injected eyes (5.2 ± 0.5 V). Interocular differences in ERGs of S334ter rats and normal mice ranged from 3% to 20% (mean% = 10 ± 6).

Our study demonstrates that intravitreal administration of rAAV.CNTF.GFP enables broad and long-term histological protection of photoreceptors in Prph2Rd2/Rd2 mice (for 8.5–9 months) and P23H and S334ter rhodopsin transgenic rats (for 6 months). Application of this vector even after retinal degeneration has commenced in the P23H and S334ter transgenic rats can still result in morphological protection of the remaining photoreceptors. This study also demonstrates that rAAV administered intravitreally is able to transduce a variety of retinal cell types in the rat, including retinal ganglion, Muller, and horizontal/amacrine cells. Intravitreal delivery may minimize exposure to the cells being protected (photoreceptors). CNTF-mediated photoreceptor protection can thus occur through delivery of the virus to other non-photoreceptor retinal cells, but the mechanism by which it exerts its effects is not clear. It is known that CNTF triggers signaling in retinal ganglion and Muller cells [15]. Binding of CNTF to its receptors in these cells specifically activates the Janus tyrosine kinase signal transducer and activators of transcription (Jak-STAT) signaling pathway [16]. It has been suggested that activation of this pathway mediates the neuroprotective effects of CNTF on mature neurons including retinal ganglion cells [16,22]. It is not known why structural protection is not accompanied by functional protection in the in vivo models. Extent of Histological Protection The retinal regions with the highest levels of CNTF–GFP expression did not correspond to those with the highest

FIG. 10. CNTF expression in the temporal retina of a 6-month-old P23H transgenic rat that received an intravitreal injection of AAV.CNTF.GFP at postnatal day 28. Note the co-localization of GFP (A, C) and CNTF (B, D) in the ganglion cells (arrowheads) and Muller cells (arrows). Sections were immunostained for CNTF and viewed under FITC (A, C) and rhodamine (B, D) filters.

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potency by the time it diffuses across the vitreous cavity. Testing the effect of CNTF concentration on photoreceptor protection should be possible with a dose-response study that would include evaluation of the effects of bilateral (that is, temporal and nasal) intravitreal injections in the same eye. Different gene mutations, stages of retinal degeneration when treatment was delivered, and species differences may all contribute to the observed variation in response to CNTF administration. Our data suggest that the rat retina has a different response to CNTF than the mouse. Different responses in different species have also been suggested by other investigators [4,24]. Perhaps the species difference that is described here is due to the ability of AAV to target multiple cell types after intravitreal injection in the rat, but only one cell type (ganglion cells) after intravitreal injection in the mouse. Transduction of Different Cell Types Intravitreal administration of rAAV led to transFIG. 11. Electroretinograms from the three animal models compared with representative normal controls. Representative normal mouse and rat dark-adapted ERGs (A). Overlaid duction of different retinal cell types in rat and responses from the two eyes of all animals studied (B). Stimulus onset is at trace onset. mouse. We expected and observed transduced Calibrations are below the responses. Histograms (C) compare average dark-adapted b- retinal ganglion cells in both species [14]. We wave amplitudes of AAV.CNTF.GFP-injected and AAV.GFP-injected eyes; error bars are also identified transduced Muller cells in adult SEM. P values, upper right corner of each comparison. rats and mice. We further verified this finding by testing AAV transduction characteristics of immortalized rat Muller cells (the rMc-1 cell line, provided by Vijay P. Sarthy, Northwestern University, extent of protection. In the Prph2Rd2/Rd2 model, photoreceptors in both hemispheres showed some protection Chicago, IL) [25]. At 36 hours after infection with compared with those in the contralateral control eye, AAV.GFP, ~ 20% of Muller cells showed GFP fluorescence although we observed less protection in the injected tem- (data not shown). The ability of rAAV to transduce Muller poral retina. However, we only noted photoreceptor pro- cells indicates that these cells have AAV receptors. tection in the nasal hemisphere in P23H and S334ter rats; Indeed, rat Muller cells have been found to express fibroblast growth factor receptor-1 (FGFR1), a receptor the photoreceptors had degenerated in the AAV.CNTF.GFP exposed temporal retinas to an extent similar to that of the for AAV-2 infection [26], in their radial fibers [27]. In contralateral control eyes. Interpretation of these effects addition, the anatomical location of Muller cells facilimust be speculative. If we assume that this difference was tates their physical contact with AAV after intravitreal not due to protection of the entire retina of the injection. Muller cells span nearly the full thickness of AAV.CNTF.GFP-injected eye with selective toxicity of the retina. Their end feet form the internal limiting CNTF on the temporal hemisphere, then how can we membrane (ILM) which borders the vitreous. The ILM contains heparin sulfate proteoglycan [28], a co-receptor explain a temporal intravitreal injection of AAV.CNTF.GFP leading to protection in the opposite (nasal) hemisphere for AAV-2 [29]. These features may make glial cells of the retina? First, the concentration of CNTF may be uniquely suited to AAV infection. Indeed, rAAV is capable of targeting other glial cells in vitro, such as oligocritical in terms of achieving protective effects. We demonstrated that transgene expression levels after intravitreal dendrocytes and astrocytes [30,31]. The search for an injection are highest in the region of the retina (temporal ideal target to produce CNTF will be a critical issue in the aspect) closest to the site of needle entry. As CNTF diffuses application of this compound for treatment of retinal away from the transduced temporal retina, a concentration degeneration. Although we observed transduced Muller cells in adult gradient may develop across the vitreous cavity. The presumed lower concentration of CNTF in the nasal portion Prph2Rd2/Rd2 mice and rhodopsin transgenic rats, we did not of the eye may be optimal for the therapeutic effect. This see the same phenomenon in neonatal Prph2Rd2/Rd2 aniconcentration gradient of CNTF may be reinforced due to mals. The reasons for this discrepancy are not clear. the short half-life of this molecule (about 1.5 min) [23]. Possible explanations would be that Muller cells in the CNTF produced by the temporal retina may have reduced neonatal Prph2Rd2/Rd2 mice have not yet developed AAV

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receptors. Indeed, Muller cells do not become fully differentiated until postnatal days 7–10 in mouse and rat [32–34]. Experiments in progress aim to determine whether differential expression of AAV receptors in Muller cells occurs during retinal development. Correlation between Gene Transfer and Photoreceptor Protection Our study intended to achieve a widespread transduction of retinal ganglion cells by using intravitreal injection. Although the transduction area is larger than that achieved by the subretinal approach [13], cell transduction, as indicated by GFP and CNTF expression, does not occur uniformly over the entire retina. Instead, it occurs in a gradient in which maximum expression is observed in the temporal half of the retina, where injections were made. The gradient-like nature of the expression may be due to the viscous nature of the vitreous humor, which would limit diffusion of the viral particles. If the virus has a short half-life, transduction would be observed only in the region maximally exposed to the virus. Consistent with this possibility, rapid binding of rAAV to neurons has been seen as early as 6 minutes after injection into the brain [35]. Differences in the extent of rescue observed in the subretinal study [13] compared with the present study may be due to differences in cell types transduced by the different surgical approaches and/or differences in the strains of animals/species. Subretinal injection of AAV leads to photoreceptor and RPE transduction; it is likely that CNTF produced by these cells activates a photoreceptor survival mechanism. In a previous study, intravitreal injection of CNTF delivered by means of recombinant adenovirus at P21 reduced photoreceptor loss and increased the amplitude of scotopic ERGs in Prph2Rd2/Rd2 mice [8]. Our study using AAV–CNTF revealed similar histological protection of photoreceptors, but no significant improvement in either scotopic or photopic ERGs. This discrepancy may be due to the differences in the vectors and their cellular specificity, the amount of time lapsed between delivery, and the assay, stability, and level of transgene expression. It is known that transgene expression mediated by adenovirus is transient due to a cell-mediated immune response [36]. Improved ERG responses in Ad-CNTF injected eyes in the previous study [8] may be attributed to regeneration of outer segments, although this effect lasts only 14 days. We did not observe this phenomenon in frozen or plastic-embedded sections from experimental or control eyes of 8- to 9-month-old Prph2Rd2/Rd2 mice. The reasons for this difference are not known, but may relate to the age of the animals or the choice of vectors. Discordant Results from Morphology and ERG Reconciliation of morphological and physiological results in this study, especially in the transgenic rats, is difficult.

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For the Prph2Rd2/Rd2 mice, amelioration of the abnormality in photoreceptor outer segment development in this condition would not be expected from treatment with CNTF; we did not observe ERG differences between treated and control eyes. The presence of ERGs in some animals even at late disease stages, such as we found, has been attributed to phototransduction occurring in the plasma membrane surrounding the cilia [37]. Transgenic rat ERGs show greater dysfunction under dark- and lightadapted conditions in the rAAV.CNTF.GFP-injected eyes compared with the rAAV.GFP-injected eyes. One possibility is that the ERG b-wave abnormalities (a-waves were not recordable due to severity of disease expression) in the rAAV.CNTF.GFP injected eyes resulted from a retinawide effect by CNTF on the b-wave generators, such as inner retinal neurons or Muller cells. This mechanism could be masking the positive physiological impact of photoreceptor protection. The suggestion has been made that Muller cells and inner retinal neurons are part of an indirect mediation of photoreceptor protection with CNTF [15,38], and the present study showed Muller cells were transduced in the rats. Another possibility is that the b-wave abnormalities are regional, like the ONL protection, but this morphologically rescued region is physiologically silent for an unknown reason, leading to only part of the retina responding in rAAV.CNTF.GFP-injected eyes. Again, we could invoke an effect by CNTF on inner retinal function or Muller cell contributions to the bwave or postulate that CNTF directly or indirectly inhibited photoreceptor function. Have there been other observations suggesting relative dysfunction in eyes treated with CNTF, whether as injections of the neurocytokine into the eye, or via other modes of delivery? A recent study in a mouse model of retinal degeneration with the P216L Prph2 mutation similarly reports unexplained functional loss despite structural preservation after intraocular injection of AAV–CNTF [39]. The literature describing the effects of another neurotrophic factor, basic fibroblast growth factor (FGF-2) on the ERG, is provocative. Intravitreal FGF-2 can reduce the ERG b-wave in wild-type rats [40]. AAV-mediated delivery of FGF-2 to S334ter heterozygous rhodopsin transgenic rats provided evidence of photoreceptor protection by histology, but ERGs did not show comparable protection [41]. Administration of CNTF may cause upregulation of secondary factors, such as FGF-2, that could be toxic to photoreceptors or other retinal cells. Although it is not known whether FGF-2 and CNTF have similar effects on ERGs, CNTF increases FGF-2 release from RPE [42]. Possible Side Effects of CNTF on Retinal Ganglion Cells One concern was whether there were any untoward effects on ganglion cells from expression of exogenous CNTF. Reports indicated that CNTF can promote neurite growth in some neuronal populations in vitro, including retinal ganglion cells [43,44]. Intravitreal administration also

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promotes the survival of RGC and regrowth of their axons after transection of the optic nerve [22,45]. Although we did not quantitatively analyze the length of RGC dendrites, we did not observe gross changes in morphology of retinal ganglion cells in our AAV-CNTF injected eyes compared with those in AAV.GFP-injected eyes. Another concern might be that CNTF would be transported from the retina to regions of the brain, mirroring the normal neuroanatomical pathways taken by ganglion cell axons. Our lab previously demonstrated high levels of reporter gene expression in optic nerve and brain after intravitreal injection of AAV–GFP [14]. Finally, CNTF receptors are localized in RGC [46] and binding of CNTF may therefore trigger unnecessary signaling. The search for an ideal target to produce CNTF will be a critical issue in the application of this compound for treatment of retinal degeneration. Studies are in progress in our laboratory to determine whether Muller cell specific targeting of CNTF will be sufficient to achieve photoreceptor rescue (and bypass a potential risk of damage to the CNS after ganglion cell transduction). In summary, rAAV.CNTF.GFP seems to be an effective vector for delaying photoreceptor degeneration as assessed histologically. CNTF-mediated protection occurs even after photoreceptors have begun to degenerate. However, the extent of protection varies between different animal models and the route (subretinal versus intravitreal) of virus delivery. There is also the unsolved problem of accompanying retinal dysfunction. An understanding of the mechanisms by which CNTF exerts its protective effects on photoreceptors will be important in optimizing its use in treatment strategies and determining whether virus-mediated delivery of CNTF could be ultimately applied to humans.

MATERIALS

AND

METHODS

rAAV vector construction and production. We constructed vectors and prepared viruses as described [13]. The CNTF virus (rAAV.CNTF.GFP) carries two transgenes: a gene encoding a secretable form of mouse CNTF [47] under the transcriptional control of the immediate early promoter from CMV and a marker gene encoding an enhanced form of GFP linked by internal ribosomal entry site (IRES). Recombinant AAV was produced by transfecting 293 cells with pAAV.CNTF.EGFP along with a helper plasmid (pAd.Help.Rep/Cap) that encodes adenovirus (E2a, E4, and VA RNA) and AAV (Rep and Cap) genes, and purified through a gradient of cesium chloride. The titer of the rAAV.CNTF.GFP virus was 2  1012 particles/ml. This virus was extensively characterized in vitro before injection into animals. Transduction of 293 cells with these viruses led to simultaneous expression of CNTF and GFP; and the CNTF product was able to promote survival of chick dorsal root ganglion neurons [13]. rAAV.GFP, used as a control in this study, was produced by cotransfection with AAV plasmids and infection with Ad-LacZ and characterized previously in our laboratory [48]. The transgene cassette consisted of EGFP under the control of the CMV promoter. The titer of this virus was 1.5  1012 particles/ml. Purified rAAV-GFP was tested for contamination of adenovirus and wild-type AAV, as described [48]. There was no evidence of adenoviral contamination as measured by histochemical assay of -galactosidase (which reveals the presence of LacZ in helper virus). Rep, reflecting the presence of replication-competent AAV, was present at a level of approximately 0.1%, as assayed by western blot analyses [48].

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Animals. Prph2Rd2/Rd2 mice and P23H line 1 and S334ter line 4 mutant rhodopsin transgenic rats were kindly provided by Richard Sidman (Tufts University School of Medicine) and Matthew LaVail (University of California, San Francisco). They were maintained as homozygotes. All procedures involving these animals were performed in accordance with the guidelines of the University of Pennsylvania for animal research. Intravitreal injections. The 3- to 5-day-old Prph2Rd2/Rd2 mice (n = 16) were injected intravitreally using a transcleral transchoroidal approach as described [14]. Briefly, after eyelid separation and conjunctival peritomy, a 33-gauge cannula was inserted through the pars plana and secured into the intravitreal cavity. Care was taken to avoid damage to the lens. One eye was injected with rAAV.CNTF.GFP, while the contralateral eye received either rAAV.GFP, a sham injection, or was left untouched. Viral solution (1 l; range of 0.5–2) was administered. Intravitreal injections were also carried out in a limited number of adult Prph2Rd2/Rd2 mice (age = 6 weeks). For the rat studies, intravitreal injections were carried out at postnatal days 28–35 in the P23H (n = 9) and S334ter (n = 7) rhodopsin transgenic rats. At this age, approximately 20% of the photoreceptors have already degenerated [20,21]. AAV.CNTF.GFP solution (2 l; range of 2–3) was injected into one eye, while the same volume of AAV.GFP solution was injected into the contralateral eye. Alternatively, sham injections were performed as control. After injection, Pred-G ointment (Allergan, Irvine, CA) was applied topically to prevent corneal desiccation and inflammation. All injections were performed under a dissecting microscope. Tissue processing. Animals were sacrificed by CO2 asphyxiation at the age of 6 months (rhodopsin transgenic rats) and 8.5–9 months (Prph2Rd2/Rd2). Before enucleation eyes were marked by cautery at the limbus in the temporal quadrant. This mark served as a reference point for orientation of the eye during embedding. Eyes were fixed in 4% paraformaldehyde and 0.5% glutaraldehyde, and cryoprotected in PBS containing 20% and 30% sucrose. Eyes were then embedded in tissue freezing medium (Triangle Biomedical Sci., Durham, NC) and sectioned at –20C in the horizontal meridian using a cryostat microtome (10 m). Sections were coverslipped with Vectorshield containing DAPI or propidium iodine (Vector Lab) which stains nuclei only. Some Prph2Rd2/Rd2 eyes were also processed for plastic embedding according to the manufacturer’s protocol (Polyscience Inc., Warington, PA), and sections (1m) were cut using a Leica ultra-microtome (Leica Microsystems Inc., Wetzlar, Germany). Morphometric analysis of photoreceptor layers. Morphometric analysis of the ONL was performed with a Leica microscope (Leica Microsystems, Inc.) under epifluorescence illumination. Images were captured with a Hamamatsu digital camera and Openlab 2.2 image analysis software (Improvision, Inc., Boston, MA). The measurements were taken in both the temporal and nasal halves of the retinas. For measurements of ONL thickness, three defined regions were selected in each hemisphere under a 10 objective. Each was separated by a distance of 200 m (Prph2Rd2/Rd2) and 400 m (rats) from the ora serrata and optic nerve head, and the third at the midpoint between these. Three measurements were taken for each region in 3–5 consecutive sections under a 40 objective with a DAPI filter. The mean of these values was used as the estimate of ONL thickness. GFP fluorescence was evaluated simultaneously in these sections under an FITC filter. To confirm the measurements of ONL thickness, the numbers of rows of nuclei in the vertical column of the ONL were counted at the same areas, again making multiple counts in multiple consecutive sections. The mean of these values was used as the estimate of photoreceptor nuclei rows in each half of the retina. Measurements of ONL thickness and the number of rows of photoreceptor nuclei have been shown to reproducibly and accurately quantify the photoreceptor population [49]. The data were processed for statistical significance using the paired Student’s t test. Immunohistochemistry for CNTF, GFAP, and PKC. Frozen retinal sections were sequentially incubated in 5% blocking serum in 0.15 M phosphate buffer (30 min), in primary antibody (chick anti-rat CNTF, 1:100, overnight at 4C (Promega); or rabbit anti-human GFAP, 1:100, 2 h at RT (Dako); or monoclonal anti-PKC, 1:200 2 h at RT (Sigma)), in secondary antibody (biotinylated goat anti-chick IgG or biotinylated anti-rabbit IgG, 1:200, biotinylated horse anti-mouse IgG 1:200, Jackson ImmunoRes Labs) for 2 h at RT, and in avidin-rhodamine (Vector Labs, Burlingame, CA) for

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1 h. Colocalization of GFP with CNTF, GFAP, or PKC was examined using epifluorescent illumination with a dual filter allowing covisualization of FITC and rhodamine. The specificity of immunostaining for CNTF and GFAP was verified by omitting primary antibodies. No immunoreactivity was observed in retinal sections subjected to staining procedures in which CNTF, GFAP, and PKC antiserum were omitted. Electroretinography. Bilateral simultaneous full-field ERGs were carried out in a subset of the animals in this study: Prph2Rd2/Rd2 mice (n = 6; age = 9 months); P23H rats (n = 7; age 6 months) and S334ter rats (n = 5; age 6 months). Untreated S334ter transgenic rats (n = 3; age 2 months) and normal mice (n = 11; age range, 2–8 months) served as controls for interocular differences in ERGs. ERGs were recorded using a custom-built ganzfeld, a computer-based system (EPIC-XL, LKC Technologies, Inc., Gaithersburg, MD) and specially made contact lens electrodes (Hansen Ophthalmic Development Lab, Iowa City, IA) [50]. Animals were darkadapted for > 12 h and anesthetized with intramuscular injections of ketamine HCl (60–75 mg/kg) and xylazine (5 mg/kg). Dark-adapted ERGs were elicited with blue flash stimuli (–0.3 log scot-cd.s.m –2); 2–10 responses were averaged with a 15-s interstimulus interval. Flicker ERGs were recorded in the light-adapted state (> 5 min) to a 15 Hz white stimulus (0.4 log cd.s.m–2); 50–100 responses were averaged. Dark-adapted ERGs were measured conventionally for b-wave amplitude and flicker ERGs for peak-to-peak amplitude.

ACKNOWLEDGMENTS We thank Artur V. Cideciyan for critical advice throughout this project and Jiancheng Huang, Elaine deCastro, Erica Dale, and Jessica Emmons (all at the University of Pennsylvania) for help with data analyses. This study was supported by 1F32 EY07073 (F.-Q.L.), NIH R01 EY10820 and EY12156 (J.B.), NIH R01 EY05627 and EY13385 (S.G.J.), the Foundation Fighting Blindness (J.B. and S.G.J.), the Lois Pope LIFE Foundation (J.B.), The William and Mary Greve International Research Scholar Award (J.B.) and Senior Scientific Investigator Award (S.G.J.) of Research to Prevent Blindness, Inc., the Ruth and Milton Steinbach Fund (J.B.), the Paul and Evanina Mackall Trust, and the F. M. Kirby Foundation. RECEIVED FOR PUBLICATION MAY 8; ACCEPTED AUGUST 27, 2001.

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