doi:10.1006/mthe.2000.0252, available online at http://www.idealibrary.com on IDEAL
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AAV-Mediated Delivery of Ciliary Neurotrophic Factor Prolongs Photoreceptor Survival in the Rhodopsin Knockout Mouse Fong-Qi Liang, Nadine S. Dejneka, Daniel R. Cohen, Natalia V. Krasnoperova,* Janis Lem,* Albert M. Maguire, Lorita Dudus,† Krishna J. Fisher,† and Jean Bennett1 F. M. Kirby Center for Molecular Ophthalmology, Department of Ophthalmology, Scheie Eye Institute, University of Pennsylvania, Philadelphia, Pennsylvania 19104 *Department of Ophthalmology, New England Eye Center, Boston, Massachusetts 02111 † Department of Pathology, Tulane University Medical Center, New Orleans, Louisiana 70112 Received for publication November 14, 2000; accepted in revised form December 21, 2000
Retinitis pigmentosa (RP), an inherited retinal degenerative disease causing blindness, is characterized by progressive apoptotic death of photoreceptors. Therapeutic modification of photoreceptor apoptosis may provide an effective therapy for this disorder. Ciliary neurotrophic factor (CNTF) has been shown to promote survival of a number of different neuronal cell types, including photoreceptors. The present study aimed to test whether adeno-associated virus (AAV)-mediated delivery of the gene encoding CNTF delays photoreceptor death in the rhodopsin knockout (opsinⴚ/ⴚ) mouse, an animal model of RP. The vector was made to express a secretable form of CNTF in tandem with a marker GFP. Cultured 293 cells transduced with this virus expressed both CNTF and GFP. The conditioned media from such cells supported the survival of chick dorsal root ganglion neurons in the same manner as recombinant CNTF. Subretinal administration of this virus led to efficient transduction of photoreceptors as indicated by GFP fluorescence and CNTF immunostaining. Histologic examination showed significant photoreceptor preservation in the injected quadrant of the retina. This protection lasted through termination of the experiment (3 months). AAV-mediated delivery of CNTF may have implications for the treatment of human retinal degeneration. Key Words: CNTF; AAV; photoreceptor; apoptosis; retinal degeneration; gene therapy; neurotrophic factor.
INTRODUCTION Retinitis pigmentosa (RP) is a group of inherited retinal degenerative diseases causing blindness. Currently there are no cures for any form of RP. The visual deficits in RP are caused by a progressive degeneration of retinal photoreceptor cells. Photoreceptor degeneration can be triggered by mutations in any of a variety of genes (1). Although the mechanism by which these genetic insults lead to photoreceptor death is still not clear, the common end result is apoptosis (2– 6). The rhodopsin knockout (opsin⫺/⫺) mouse is a wellcharacterized animal model of photoreceptor degenera1 To whom correspondence and reprint requests should be addressed at the Scheie Eye Institute, University of Pennsylvania, 310 Stellar–Chance Laboratories, 422 Curie Boulevard, Philadelphia, PA 19104-6069. Fax: (215) 573-7155. E-mail:
[email protected]. upenn.edu.
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tion, in which both opsin alleles have been disrupted (7). The retinas in these mice initially develop normally; however, the outer segments fail to form in rod photoreceptors. By the age of 1 month, 10 –15% of rod nuclei are lost, and by 3 months over 90% are lost. This leaves only a single, fragmented row of nuclei composed exclusively of cones. This model thus allows rapid testing of therapies for human RP. The animal model is thought to most closely mimic the disease manifested in humans with autosomal recessive rhodopsin null mutations (8). In recent years, several trophic factors, including ciliary neurotrophic factor (CNTF), have been shown to promote photoreceptor survival in animal models with retinal degeneration (9 –16). CNTF is expressed in both developing and mature retina in the rat and is predominantly localized in Muller cells (17). In order to exert its function, CNTF must bind to its specific receptor, the ␣ subunit of the CNTF receptor complex, which is located in Muller,
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FIG. 1. Schematic illustration of the transgene cassette of pCMV.CNTF.GFP. The black arrows represent inverted terminal repeats.
horizontal, amacrine, and ganglion cells. The receptor is not found in photoreceptors (17–20). The protective effects of CNTF on photoreceptor degeneration are intriguing. A single bolus injection of a modified recombinant CNTF protein into the vitreous of any of a variety of mouse models, including the rd/rd, Q344ter rhodopsin transgenic, and nervous mutant mouse, led to short-term (7–10 days) rescue of photoreceptors (10). The CNTF protein has a short half-life (1.5 min) (21), and so it was thought that a delivery system which would increase its availability over longer time periods might be more effective. When CNTF was delivered by recombinant adenovirus, it delayed photoreceptor degeneration in both the rd/rd mouse and the rds/rds mouse (22, 23). Recent reports also indicate that recombinant adeno-associated virus (rAAV)- or CNTF-producing cells can be harnessed for similar therapeutic effects in the S344ter and the P23H rhodopsin transgenic rats and the rcd1 dog, respectively (14, 15, 24). These findings suggest that a continual supply of CNTF can prolong the life of photoreceptors. One challenge with such studies is that CNTF expression cannot be monitored in vivo and that it is difficult to identify CNTF-expressing cells histologically. In the present study, we have used a reporter gene, GFP, as a marker for CNTF-expressing cells. This was made possible by the insertion of an internal ribosome entry site (IRES) sequence between CNTF and the GFP open reading frame. The bicistronic transgene cassette was delivered by rAAV, which targets photoreceptors efficiently and results in stable transgene expression (25–28). Here we demonstrated a rescue effect of CNTF in retinal regions that possess GFP. It should be possible to use this paradigm to assess the ability of CNTF to promote photoreceptor survival in a variety of animal models of retinal degeneration.
METHODS rAAV vector construction and production. A plasmid that encodes a secretable form of murine CNTF (pCNTF-chim5) (29) was kindly provided by Dr. Michael Sendtner (University of Wurtzburg, Germany). The CNTF cDNA was released by restriction digestion and inserted into the multiple cloning site of plasmid pIRES2-EGFP (Clontech, Palo Alto, CA). This cloning strategy placed the CNTF cDNA under the transcriptional control of the immediate early promoter from cytomegalovirus (CMV). The resulting transgene cassette also expressed the marker EGFP due to an IRES element positioned between the CNTF and the EGFP open-reading-frames. The bicistronic cassette was subcloned into a derivative of AAV plasmid psub201 (30) that was deleted of all viral genes, yielding plasmid pAAV.CNTF-EGFP (Fig. 1). 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. Cells were harvested 72 h posttransfection and virus was purified as described (31). Purified rAAV.CNTF-EGFP vector was titered
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by a PCR-based assay for virion DNA particles (31). The titer was 2 ⫻ 1012 particles/ml. rAAV.GFP, used as a control in this study, was produced and characterized previously in our laboratory (26, 28). The transgene cassette consisted of EGFP under the control of the CMV promoter. The titer of this virus was 1.5 ⫻ 1012 particles/ml. Analysis of CNTF expression by rAAV.CNTF.GFP in vitro. The 293 cells were cultured in six-well culture plates (Becton–Dickinson Labware, Franklin Lakes, NJ) with DMEM (Gibco BRL, Gaithersburg, MD) supplemented with 10% fetal bovine serum. When cells reached approximately 80% confluence, they were infected with either rAAV.CNTF.GFP (2 l/well, 4 ⫻ 109 particles) or rAAV.GFP (2 l/well, 3 ⫻ 109 particles) as control. After 48 h, the medium was collected and cells were harvested. The presence of CNTF protein in the conditioned medium and protein extracts from cells was determined by ELISA and Western blot analysis. The cells were also fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS) for immunostaining of CNTF. Western blotting. The conditioned medium from the transduced 293 cells was filtered with a Centricon YM-50 filter (Millipore, Bedford, MA) to enrich ⬍50-kDa proteins (CNTF monomer, 23 kDa; dimer, 46 kDa). Protein concentrations in the 293 cell lysates and conditioned media were determined by the Bio-Rad protein assay (Bio-Rad, Hercules, CA). Protein samples (36 –250 g) and recombinant rat CNTF (rrCNTF; 100 ng; Promega, Madison, WI) were solubilized in SDS-loading buffer and subjected to SDS–polyacrylamide (15%) gel electrophoresis (60 mA for 1 h). Proteins were electroblotted onto nitrocellulose (20 V for 1 h) and the blot was sequentially incubated in primary antibody (1:500 chick anti-rat recombinant CNTF; Promega, Madison, WI) and secondary antibody, a horseradish peroxidase-conjugated rabbit anti-chicken IgY (1:5000; Jackson ImmunoResearch Laboratory, West Grove, PA). The ECL kit (Amersham, Austin, TX) was used to generate a chemiluminescent signal. CNTF ELISA. CNTF levels in the conditioned media and opsin⫺/⫺ retinal extracts were determined using quantitative sandwich ELISA. Retinas were dissected from the 3-month-old opsin⫺/⫺ mice that had received subretinal injections of rAAV.CNTF.GFP and rAAV.GFP at postnatal day 3 (see below) and frozen individually in dry ice. Each retina was homogenized in ice-cold 10 mM phosphate buffer (pH 7.4) containing 30 mM NaCl, 0.1% Tween 20, 0.1% bovine serum albumin (Sigma), and protease inhibitors (Sigma). The suspensions were then centrifuged at 15,000g for 20 min at 4°C, and the supernatant was collected. Protein concentration of each sample was measured by the Bio-Rad protein assay. Flat-bottom 96-well microplates (Costar, Cambridge, MA) were coated overnight at 4°C with 100 l of monoclonal rabbit anti-CNTF antibody (2 g/ml; R&D Systems, Minneapolis, MN) diluted in 0.025 M sodium carbonate buffer (pH 9.7). With interceding washes (50 mM Tris-buffered saline (TBS), pH 7.4), the plates were subjected to sequential 2-h incubations at room temperature with blocking solution (1% BSA in TBS), triplicate aliquots of conditioned medium, protein extracts from opsin⫺/⫺ retinas or rrCNTF (0.01–2 ng/well; Promega), polyclonal chicken antiCNTF antibody (1 g/ml; Promega), and HRP-conjugated rabbit antichicken IgY (1:5000; Jackson ImmunoResearch). HRP activity was detected using 3,3⬘,5,5⬘-tetram-ethylbenzidine (Promega) as the color substrate. After a 10 –20 min incubation, color reaction was stopped by adding an equal volume of 2 N HCl. Absorbance at 450 nm was measured using a Victor plate reader (Perkin–Elmer Wallac, Inc., Gaithersburg, MD). Using serial dilutions of known amounts of rrCNTF, this color reaction yielded a linear standard curve from 0.0625 to 1 ng. The CNTF levels in the samples were quantified within the linear range of the standard curve and normalized for the total protein (g) that was assayed in each retina. The Student t test was used to analyze the differences in the CNTF levels between the rAAV.CNTF.GFP- and the rAAV.GFP-injected eyes. Bioassays of CNTF produced by rAAV.CNTF.GFP. Production of biologically active CNTF was identified using cultured dorsal root ganglion neurons. Ganglia were dissected from E8 –9 chick embryos (N ⫽ 8) and cultured as described by Barde et al. (32). After trypsinization, the cell suspension was plated in DMEM supplemented with 10% fetal bovine serum for approximately 3 h in a 60-mm plastic dish (Sarstedt, Inc., Newton, NC). During this period, most of the nonneuronal cells adhered to the plastic surface, while the neuronal cells remained in suspension. The neurons were resuspended and seeded at a density of ⬃10,000 cells/ MOLECULAR THERAPY Vol. 3, No. 2, February 2001 Copyright © The American Society of Gene Therapy
ARTICLE well in 24-well plates precoated with polylysine/laminin (Sigma). They were grown in DMEM with 10% FBS that was supplemented with 50% (v/v) AAV.CNTF.GFP- or AAV.GFP-conditioned medium or a premixed solution of CNTF neutralizing antibody (15 g/ml; R&D Systems) and rAAV.CNTF.GFP-conditioned medium. Positive control wells were supplemented with 10 ng/ml rrCNTF. After 48 h, cultures were fixed, and the numbers of surviving neurons were counted under a phase-contrast microscope. Five fields were randomly selected from each well for estimation of surviving neurons. Only phase-bright neurons bearing processes were included in the evaluation of neuronal survival. The treatment effect on survival was analyzed using ANOVA. Animals and surgical procedures. The opsin⫺/⫺ mice were maintained in a temperature-controlled room under a standard 12-h photoperiod (LD 12: 12; lights on at 0600 h). Throughout the study, animals were provided with access to food and water ad libitum. All procedures involving these animals were performed in accordance with the ARVO statement for the use of animals in ophthalmic and vision research and the guidelines of the University of Pennsylvania for animal research. Animals (2–5 days of age, N ⫽ 32) were injected subretinally using a transscleral transchoroidal approach as described previously (26, 33). This resulted in delivery of material to the temporal retina. Briefly, after eyelid separation, a 33-gauge cannula was inserted and secured into the subretinal space at the temporal peripheral retina following conjunctival peritomy. One eye was injected with rAAV.CNTF.GFP, while the contralateral eye received rAAV.GFP or a sham injection. The amount of transfection solution injected was 0.5–1 l (1–2 ⫻ 109 particles of rAAV), thereby raising a dome-shaped retinal detachment. After injection, Pred-G ointment (Allergan, Irvine, CA) was applied topically to prevent corneal desiccation and inflammation. All injections were monitored by direct visualization through an operating microscope. Animals were sacrificed by CO2 asphyxiation at the age of 3 months, except that 5 animals were terminated at the age of 4 weeks to examine GFP expression in the retina. Indirect ophthalmoscopy. Prior to sacrifice, animals were examined with indirect ophthalmoscopy. This was performed with a 90-diopter lens following pupil dilation with topically applied 0.5% tropicamide as described previously (26, 34). Photographs were taken using a Kowa camera (Keeler Instruments, Broomall, PA). Qualitative assessment was performed by an observer who was masked as to the treatment administered. Fundus appearances in the rAAV.CNTF.GFP-treated eyes were compared with those injected with rAAV.GFP and untouched eyes. Immunocytochemistry for CNTF. To examine CNTF expression in the retina and transduced 293 cells, paraformaldehyde-fixed 293 cells and cryostat retinal sections were sequentially incubated in 5% blocking serum in 0.15 M phosphate buffer (20 min), in chick anti-rat CNTF antibody (1:100; Promega) for 12–16 h at 4°C, in biotinylated goat anti-chick IgG (1:100; Jackson ImmunoResearch) for 2–3 h at room temperature, and in avidin D–rhodamine or avidin– biotin–alkaline phosphatase complex (Vector Laboratories, Burlingame, CA) for 1 h. Immunoreactive elements in the retinal sections were then identified using a chromogen solution containing VectaRed (Vector Laboratories) so as to yield a fluorescent red reaction product. Colocalization of GFP and CNTF was studied using epifluorescence illumination with a dual filter allowing covisualization of FITC and rhodamine. The specificity of immunostaining for CNTF was verified by omitting primary antibody and preabsorption controls. No immunoreactivity was observed in retinal sections subjected to staining procedures in which the CNTF antiserum was omitted or was preincubated with recombinant rat CNTF. Morphometric analysis of photoreceptor layers. Prior to enucleation, eyes were marked by cautery at the limbus in the temporal quadrant. This mark served as a reference point to orient the eye during embedding. Eyes were fixed in 4% paraformaldehyde and cryoprotected in PBS containing 20 and 30% sucrose. Eyes were then embedded in tissue freezing medium (Triangle Biomedical Science, Durham, NC) and sectioned at ⫺20°C in the horizontal meridian using a cryostat microtome (10 –12 m). Sections were coverslipped with Vectorshield containing DAPI (Vector Laboratories). Morphometric analysis of the outer nuclear layer (ONL) was performed with a Leica microscope (Leica Microsystems, Wetzlar, Germany) 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 MOLECULAR THERAPY Vol. 3, No. 2, February 2001 Copyright © The American Society of Gene Therapy
temporal and the nasal halves of the rAAV.CNTF.GFP-injected retinas and only in the temporal halves of rAAV.GFP- or sham-injected retinas. For measurements of ONL thickness, three defined regions, separated by a distance of 200 –300 m, were selected from each half of the retina under low magnification (Fig. 5). The distance of each region to the ora serata was also measured with an eyepiece micrometer to serve as a reference point for other measurements. Three measurements were taken for each location in three to five consecutive sections under a 40⫻ objective. The mean of these values was used as the estimate of ONL thickness. GFP fluorescence was evaluated simultaneously in these sections. To confirm the measurements of ONL thickness, the number of rows of photoreceptor nuclei was counted at the same spots, 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 (35). All measurements were performed independently by two investigators. The difference in results between the two observers was less than 10%, and the mean was taken as the final result. The data were processed for statistical significance using ANOVA.
RESULTS Expression of CNTF and GFP by 293 Cells Transduced with rAAV.CNTF.GFP To determine whether transduction with rAAV.CNTF. GFP can cause cells to produce CNTF and GFP, 293 cells were infected with this virus (4 ⫻ 109 particles/well). Within 15 h, GFP fluorescence was apparent in these cells. It became brighter, and the number of GFP-fluorescent cells increased over the next 24 h. After 48-h transfection, approximately 15% of cells showed GFP fluorescence (Fig. 2A). To ensure that the cells showing GFP fluorescence also expressed CNTF, immunostaining for CNTF was performed 48 h postinfection. CNTF was observed in all GFP-positive cells (Fig. 2B). Moreover, CNTF expression was also detected in some cells that lacked GFP. Approximately 40% of cells showed CNTF immunostaining. CNTF staining was not observed in the 293 cells transduced with rAAV.GFP (Fig. 2D), although many cells (⬃60%) showed GFP fluorescence (Fig. 2C). To verify that the CNTF produced was of the correct molecular size and secretable, cellular protein extracts and conditioned media were analyzed by Western blot. Mature CNTF protein was consistently detected in both protein extracts and media from cells transduced with rAAV.CNTF.GFP as prominent immunoreactive bands with the molecular sizes of 23 (monomer) and 46 kDa (dimer) (Fig. 3). Western blotting of recombinant rat CNTF, which served as a positive control, also yielded two individual protein bands corresponding to these sizes. A 46-kDa CNTF dimer was expected as it has been frequently observed in other studies (17, 36, 37). Neither protein extracts nor media from 293 cells transduced with rAAV.GFP produced proteins that cross-reacted with the anti-CNTF antibody. The CNTF protein appeared more abundant in the cell extract than in the medium (Fig. 3). This was due to the fact that the amount of protein loaded in the lane representing cell extract (250 g) was much greater than that loaded in the lane representing medium
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FIG. 2. CNTF immunostaining in cultured 293 cells infected with rAAV.CNTF.GFP (A and B) and rAAV.GFP (C and D). GFP fluorescence was apparent in cells transduced with both AAVs after 48 h (A, C). CNTF expression, however, was observed only in the rAAV.CNTF.GFP-infected cells (B) and was absent in the rAAV.GFP-infected cells (D). Arrows indicate cells expressing both GFP and CNTF.
(68 g). The presence of CNTF protein in the conditioned media from rAAV.CNTF.GFP-transduced cells was also confirmed with ELISA. Transduction of 293 cells with 4 ⫻ 109 particles resulted in 18.83 ⫾ 2.56 ng (mean ⫾ SD) CNTF protein/106 cells/24 h. CNTF levels were undetectable in the rAAV.GFP conditioned medium.
Neuroprotection of Cultured Sensory Ganglia Neurons by rAAV.CNTF.GFP-Conditioned Medium To determine if the virus-delivered CNTF is biologically active, rAAV.CNTF.GFP-conditioned medium was examined to see if it promoted the survival of cultured dorsal root ganglion neurons. The number of surviving neurons treated with rAAV.CNTF.GFP-conditioned medium was significantly greater than that treated with rAAV.GFPconditioned medium (mean ⫾ SD, 380 ⫾ 35 vs 108 ⫾ 16, P ⫽ 0.0009). Importantly, this was comparable to the number of surviving neurons treated with rrCNTF (401 ⫾ 42, P ⫽ 0.082). Addition of neutralizing CNTF antibody
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(15 g/ml) abolished the survival-promoting effects of rAAV.CNTF.GFP-conditioned medium. The number of surviving neurons treated with a mixture of rAAV.CNTF. GFP-conditioned medium and neutralizing antibody was 124 ⫾ 20, which was similar to that treated with rAAV.GFP-conditioned medium (P ⫽ 0.15).
Clinical Appearance of the rAAV.CNTF.GFP-Injected Eyes To determine if CNTF can improve retinal health, the fundi of opsin⫺/⫺ mice were examined with indirect ophthalmoscopy at the end of the experiment. A widespread pigmentary retinopathy and thinning of retinal blood vessels were observed in the rAAV.GFP-injected eyes at the age of 3 months (Fig. 4A). This was similar to the appearance of untreated age-matched opsin⫺/⫺ retinas (data not shown). In contrast, eyes treated with rAAV. CNTF.GFP had subtle pigmentary abnormalities and thicker retinal blood vessels (Fig. 4B). There was minimal inflammation in rAAV-injected eyes. MOLECULAR THERAPY Vol. 3, No. 2, February 2001 Copyright © The American Society of Gene Therapy
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FIG. 4. Fundus photographs of a 3-month-old opsin⫺/⫺ mouse injected with rAAV.GFP in one eye (A) and with AAV.CNTF.GFP in the contralateral eye (B) at postnatal day 3. Arrows indicate pigmentary deposits.
FIG. 3. Western blot analysis of CNTF expression in 293 cells infected with rAAV.CNTF.GFP and rAAV.GFP. In rAAV.CNTF.GFP-infected cells, CNTF was detected in both protein extracts (lane 3) and conditioned media (lane 5). rrCNTF (100 ng) served as a positive control (lane 1). In AAV.GFP-infected cells, CNTF was detectable neither in protein extracts (lane 2) nor in media (lane 4).
Histologic Rescue of Photoreceptors Marked rescue effects were observed in the temporal region of the retina which had been exposed to rAAV. CNTF.GFP (Fig. 5A). There was no apparent rescue in the nasal (unexposed) region of the same retina (Fig. 5B). The
rescue areas varied in size from eye to eye due to variability from injection to injection, but covered approximately 1⁄4–1⁄2 of the temporal half of the retina. The extent of rescue corresponded to the number and distribution of GFP-expressing photoreceptors. Both extent of rescue and number of GFP-expressing photoreceptors were maximal at the injection site and tapered from this site in a gradient. At the edge of the treated portion of the retina, a border can be detected between rescued and nonrescued regions (Fig. 5A). Morphometric analysis revealed significant differences in the ONL thickness and the number of rows of photoreceptor nuclei between the regions exposed and those
FIG. 6. Comparison of GFP fluorescence and histological rescue by rAAV.CNTF.GFP and rAAV.GFP in the opsin⫺/⫺ retina. Cryostat sections of a 3-month-old opsin⫺/⫺ mouse that had received a subretinal injection of rAAV.CNTF.GFP in one eye (A and B, temporal area; C and D, nasal area) and of rAAV.GFP in the contralateral eye (E and F, temporal area). Sections were viewed under DAPI (A, C, and E) and FITC (B, D, and F) filters. GFP fluorescence was observed in photoreceptors in rAAV.CNTF.GFP-injected portion of the retina (B). In contrast, GFP was absent in both the nasal retina (D) of the rAAV.CNTF.GFP-injected eye and the temporal retina of the contralateral eye (F). rpe, retinal pigment epithelium; onl, outer nuclear layer; inl, inner nuclear layer; gcl, ganglion cell layer. A, B and C, D represent the enlarged areas of Figs. 5A and 5B, respectively.
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ARTICLE point at which animals were sacrificed) and maintained throughout the experiment (3 months following injection). Note that not all photoreceptors in the retina expressed GFP. Most GFP-fluorescent cells were dispersed over a region comprising approximately 20 –30% of the temporal half of the retina. The concentration of GFPfluorescent cells was maximal in the center of the injection area (Fig. 6B). In addition to photoreceptor transduction, GFP fluorescence was variably observed in RPE cells in the temporal regions of retinas. Occasionally, a few cells in the inner nuclear layer (horizontal and/or amacrine) also expressed GFP. There was no apparent GFP expression in the nasal half of the retina of rAAV.CNTF. GFP-injected eyes (Fig. 6D). In comparison, strong GFP fluorescence was observed in rAAV.GFP-injected eyes 4 weeks following injections. Similarly, GFP was predominantly expressed in the photoreceptor cells in the injected area. However, very few photoreceptor nuclei showed GFP fluorescence at the end of experiment (3 months of age) since most photoreceptors had degenerated (Fig. 6F).
Expression of CNTF by rAAV.CNTF.GFP-Transduced Photoreceptors FIG. 5. Light micrographs of temporal (A) and nasal (B) retinas in a 3-monthold opsin⫺/⫺ mouse that had received a subretinal injection of rAAV.CNTF.GFP at the postnatal day 3. Morphometric analysis of the ONL was performed in the designated boxes in (A) and the corresponding areas in (B). Note the focal preservation of photoreceptor cells in the temporal retina (A) and a single fragmented row of photoreceptor nuclei in the nasal retina (B). ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer.
unexposed to rAAV.CNTF.GFP. Similarly, there was no evidence of rescue in the retina treated with rAAV.GFP (Table 1). The rAAV.CNTF.GFP-injected region (Fig. 6A) displayed a significantly thicker ONL compared to the noninjected portion of the same eye (P ⬍ 0.00001, Fig. 6C) and the corresponding region of the contralateral control eye (P ⬍ 0.00001, Fig. 6E). The number of rows of photoreceptors was also significantly greater in the rAAV.CNTF.GFP-injected retinas compared to the noninjected nasal retinas or the corresponding regions of the contralateral control eye (P ⬍ 0.00001). Typically, the rAAV.CNTF.GFP-injected area possessed four to five rows of photoreceptor nuclei (Fig. 6A) as opposed to one single, discontinuous row in the nasal retina of the same eye or in the contralateral eye (Figs. 6C and 6E). Although photoreceptor nuclei were preserved in rAAV.CNTF.GFP-injected areas, outer segments of photoreceptors were still absent in both experimental and control opsin⫺/⫺ retinas.
rAAV.CNTF.GFP-Mediated Gene Transfer in the Retina To examine what retinal cells have been transduced by rAAV.CNTF.GFP after subretinal administration in the opsin⫺/⫺ mice, appearance of GFP fluorescence in the cryostat retinal sections was examined with a fluorescence microscope. GFP fluorescence was observed in the photoreceptor cells 4 weeks after injections (the earliest time
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To verify whether histological photoreceptor rescue was due to CNTF production in the rAAV.CNTF.GFP-injected eyes, immunohistochemistry and ELISA were performed using a commercially available anti-rat CNTF antibody (Promega). Although CNTF staining was weak in the nerve fiber layer and the ciliary body of both rAAV. CNTF.GFP- and rAAV.GFP-injected eyes, strong CNTF immunoreactivity was observed in some photoreceptors which simultaneously expressed GFP in the rAAV.CNTF. GFP-injected eyes (data not shown), suggesting that rAAV.CNTF.GFP-transduced photoreceptors produced CNTF. Nevertheless, the number of CNTF-secreting immunopositive cells was less than that of the photoreceptors showing cytoplasmic GFP fluorescence. In contrast, there was no CNTF immunoreactivity in photoreceptors of the rAAV.GFP-injected eyes. ELISA of retinal protein extracts from a separate group of animals (N ⫽ 5) yielded similar indication of increased CNTF levels in the rAAV.CNTF. GFP-injected retinas. CNTF content in rAAV.CNTF.GFP-
TABLE 1 Comparison of ONL Thickness and Number of Rows of Photoreceptor Nuclei in Opsin⫺/⫺ Mice 3 Months after Subretinal Injections of rAAV.CNTF.GFP and rAAV.GFP Treatment rAAV.CNTF.GFP (n ⫽ 16) Injected area Noninjected area rAAV.GFP/sham (n ⫽ 16) Injected area
ONL thickness (m)
Number of rows
63.96 ⫾ 16.67* 14.62 ⫾ 2.31
4.63 ⫾ 1.94* 1.00 ⫾ 0.17
18.17 ⫾ 4.98
1.00 ⫾ 0.41
Note. Numbers represent means ⫾ SD. * P ⬍ 0.00001. MOLECULAR THERAPY Vol. 3, No. 2, February 2001 Copyright © The American Society of Gene Therapy
ARTICLE injected retinas ranged from 1.39 to 1.68 pg/g of soluble protein (mean ⫾ SD, 1.52 ⫾ 0.12). In contrast, CNTF levels were significantly lower (P ⫽ 0.0015) in rAAV.GFPinjected retinas, ranging from 0.68 to 1.13 pg/g of soluble protein (mean ⫾ SD, 0.89 ⫾ 0.18).
DISCUSSION The present results demonstrate that rAAV.CNTF.GFP is able to transduce 293 cells in vitro, allowing for simultaneous expression of both CNTF and GFP. CNTF produced by this virus is biologically active, as shown by its ability to promote the survival of chick sensory ganglion neurons in vitro. Importantly, subretinal administration of this virus leads to efficient transduction of photoreceptor cells and prolongs their survival (through at least 3 months) in the opsin⫺/⫺ mouse. Simultaneous expression of GFP by this vector served as a useful tool to identify the injected area of the retina and facilitated quantification of photoreceptor rescue. The rAAV.CNTF.GFP-mediated protection appeared to be specific for CNTF, because photoreceptor rescue was only observed in the regions of the retina where CNTF was coexpressed with GFP. Such effects were not seen in the uninjected areas of the same retina or the contralateral control eyes. In vitro studies showed a smaller number of GFP-fluorescent cells than CNTF-immunopositive 293 cells (as shown in Fig. 2). The inequality in expression of the two transgenes is likely due to the promoters used in the transgene construct. In this study, CMV was used to drive CNTF expression. This was in turn connected to an IRES– EGFP sequence. It is known that the IRES sequence is generally 5–10 times less strong than (but is often half as strong as) the initial CMV promoter in the transgene cassette (38). Thus, lower levels of GFP expression are expected in the cells transduced with the AAV.CNTF.GFP viruses. Despite the fact that GFP expression was not observed in every exposed photoreceptor, there was photoreceptor rescue over the entire rAAV.CNTF.GFP-injected portion of the opsin⫺/⫺ retina. This may be due to the fact that CNTF is secretable, allowing a bystander effect. In contrast, GFP is not secretable and has a cytoplasmic location. Secretion of CNTF seems to be important for achieving rescue, as delivery of the nonsecretable form of CNTF does not lead to rescue (Bennett et al., unpublished data). One puzzle is to determine how delivery of CNTF leads to rescue. Photoreceptor cells do not express the CNTF-specific receptor (17–19). Delivery of CNTF can, however, activate Muller cells and other cells of the neural retina (19, 20). We hypothesize that photoreceptor-mediated delivery of CNTF to Muller cells activates a separate survival-promoting activity. We are in the process of testing this hypothesis. An understanding of the mechanism(s) by which CNTF exerts its rescue effects on photoreceptors will be important in optimizing treatment strategies. Apparently, CNTF does not diffuse far beyond the region exposed by subretinal injection. In the present study, 0.5–1 l of rAAV.CNTF.GFP was administered to neonatal MOLECULAR THERAPY Vol. 3, No. 2, February 2001 Copyright © The American Society of Gene Therapy
mice, exposing approximately 20 –30% of half the retina. Local rescue has been observed in previous studies using other rescue agents with a similar delivery approach (33, 34, 39). Thus, delivery of larger volumes of transfection solution, which is likely to expose more of the retina, may be required to achieve a greater therapeutic effect. Alternatively, intravitreal injection of rAAV may be an efficient route for targeting the entire retina. This approach has been used to deliver recombinant CNTF protein and adenoviruses carrying CNTF (10, 22, 23). Unfortunately, adenovirus is rapidly cleared after subretinal injection due to a cell-mediated immune response (42). This would account for the fact that Cayouette and Gravel documented rescue after delivery of Ad-CNTF for only a 2to 3-week period (22, 23). Intravitreal delivery of rAAV results in efficient transduction of ganglion cells (40). It may be possible to harness these cells to produce CNTF, which could then diffuse to the outer retina. There are, however, potential challenges with such an approach. For example, CNTF secreted by ganglion cells may cause toxic effects to other cells bordering the vitreal cavity, such as lens epithelia. In fact, cataract and inflammatory responses have been reported in dog eyes injected with CNTF protein intravitreally (41). A second concern is that CNTF produced in retinal ganglion cells may be delivered to structures in the brain, leading to undesirable effects (40). Studies in our laboratory are under way to determine whether intravitreal delivery of rAAV.CNTF.GFP can lead to broad rescue of photoreceptors with minimal toxicity. The differences in rescue when using the different viruses and delivering them to different cavities may provide clues as to the mechanism by which CNTF rescues photoreceptor cells. Finally, it would be interesting to examine whether functional rescue has been achieved in these opsin⫺/⫺ mice. So far we have not detected rescue using full-field ERGs (data not shown; Aleman and Jacobson, personal communication). Photoreceptors of opsin⫺/⫺ mice lack rhodopsin and outer segments (7) and thus lack recordable rod (and cone) ERG responses. As we did not deliver a protein that restores function (i.e., rhodopsin), it is unlikely that physiological rescue would be observed. Nevertheless, a delay in rod photoreceptor cell death is likely to prolong the life of cone photoreceptors. In addition, it should be noted that presence of the photoreceptor cells is a prerequisite for additional treatment(s) that could correct the genetic defect. One of the most promising implications of this study is that AAV-delivered CNTF could be used to delay death of photoreceptors until a more direct repair could be made. AAV.CNTF could be used as an adjunctive treatment, could be part of a gene therapy cocktail, or could be used prior to identifying a vector that could address the specific defect. Treatment with AAV.CNTF could minimize photoreceptor cell loss until all of the diseased cells in the retina could be targeted using a vector tailored to the specific disease. A delay in degeneration induced by treatment with AAV. CNTF could also permit the cells to persist long enough
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ARTICLE that the genetic defect could be corrected by new technological approaches such as “chimeroplasty” (43). This study demonstrates that rAAV.CNTF.GFP can provide significant protection to degenerating photoreceptors in the opsin⫺/⫺ mouse. The coexpression of GFP induced by this vector allows for identification of transgene expression. Since photoreceptors die through a common apoptotic pathway in animal models of RP, it will be interesting to determine whether this vector can exert similar protective effects in other animal models. Continual supply of CNTF mediated by AAV may ultimately prove useful either alone or in combination with other treatments in human retinal degenerative diseases. ACKNOWLEDGMENTS The authors are deeply indebted to Drs. Tomas Aleman and Samuel G. Jacobson for ERG recording and reviewing the manuscript. We also thank Drs. Vibha Anand and ThucAnh Ho for technical assistance and Drs. Jonathan Raper and Richard Stone for providing chick embryos. This study was supported by NIH Training Grant DK77840 (F.Q.L.), NIH RO1 EY10820 and EY12156 (J.B.), the Foundation Fighting Blindness, the Paul and Evanina Mackall Trust, the Lois Pope LIFE Foundation, the William and Mary Greve International Research Scholar Award (Research to Prevent Blindness, Inc.), the Ruth and Ann Milton Steinbach Fund, and the F. M. Kirby Foundation.
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