The EcoRI/KpnI fragment of mouse RPE65 cDNA (GenBank Accession Number: NM_029987) was inserted into the pCI mammalian expression vector (Promega Corp., WI, USA) to produce a pCI.RPE65 subclone. A 3800 bp cassette, consisting of the RPE65 cDNA flanked by a 5' human cytomegalovirus (CMV) promoter and a 3' SV40 late polyadenylation signal sequence, was removed from pCI.RPE65 by BglII/BspHI restriction enzyme digest. This cassette was then inserted between the inverted terminal repeats of the serotype 2 rAAV plasmid pSSV9 24. The insertion was achieved by blunt end ligation of the 3800 bp CMV.RPE65 cassette with the large fragment of pSSV9 following XbaI digestion 24. The identity of the pSSV9.CMV.RPE65 vector was confirmed by restriction enzyme analysis. The expression of RPE65 protein from pSSV9.CMV.RPE65 was confirmed by western blot analysis of pSSV9.CMV.RPE65-transfected, human embryonic kidney (HEK) 293 cells using a rabbit anti-RPE65 polyclonal antibody 25.

pSSV9.CMV.RPE65, AAV helper (Ad8) and adenovirus helper plasmid DNA were co-transfected into HEK293 cells. The excision and replication of the resultant rAAV.RPE65 DNA was verified by Hirt analysis 26. Upon successful verification, cesium chloride gradient purified pSSV9.CMV.RPE65 DNA was either co-transfected with Ad8 and adenovirus helper plasmid DNA into HEK293 cells and the resulting virus (rAAV.RPE65) purified by cesium chloride gradient density as previously described 24, or was sent to the Vector Core Facility (University of North Carolina, NC, USA) for large-scale virus production, where the virus (rAAV.RPE65.1) was purified using iodixanol gradient followed by heparin-affinity chromatography according to published methods 27. The titers of rAAV.RPE65 and rAAV.RPE65.1 were both 6 × 10¹³ particles/ml.

All procedures were approved by the University of Western Australia Animal Experimentation Ethics Committee and were in compliance with the Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Vision Research. Mice were housed in cages in rooms maintained at constant temperature (22°C) and humidity (50%) and with a 12:12 hr light-dark cycle. Food (Glen Forest Rodent Chow, Australia) and water were given ad libitum.

Rpe65^-/- mice were anesthetized by intraperitoneal injection of ketamine (30 mg/kg) and xylazine (8 mg/kg), and their pupils dilated with topical application of a mixture containing 2.5% phenylephrine hydrochloride and 1% tropicamide (Alcon, Australia). The conjunctiva was cut and the sclera exposed. A shelving puncture of the sclera was made with a 30-gauge needle. A 32-gauge needle attached to a 5 μl Hamilton syringe was passed tangentially through the site of the sclera puncture under an operating microscope. A 1 μl solution containing 6 × 10¹⁰ particles of rAAV.RPE65 or rAAV.RPE65.1 was then delivered into the subretinal space of the mouse eye, the diameter of which is about 3.5 mm. Successful delivery of virus into the subretinal space was confirmed by the presence of a 1 to 1.3 mm diameter circular bleb when examined by indirect ophthalmoscopy (approximately 30% of the retinal area). The needle was kept in the subretinal space for 1 min, and then withdrawn gently. Finally, a layer of antibiotic ointment was applied to the injected eye. Additional Rpe65^-/- mice were injected with 1 μl of the control construct rAAV.GFP. All mice used in this study were injected upon reaching maturity, at 3 weeks of age.

rAAV.RPE65-injected and uninjected Rpe65^-/- mice were analyzed by electroretinography (ERG) at 1–2 mo (n = 15 rAAV.RPE65-injected, n = 10 uninjected), 7 mo (n = 12 rAAV.RPE65-injected, n = 4 uninjected) and 11 mo (n = 12 rAAV.RPE65-injected, n = 6 uninjected) post-injection. Following dark-adaptation of the mice, full-field scotopic flash ERGs were recorded. The mice were anesthetized as described earlier and maintained at 37°C with a homeothermic electric blanket. Their pupils were dilated with 0.5% tropicamide (Alcon) and the cornea was protected with carmellose sodium (Celluvisc, Allergan, Australia). The ERG was recorded between a platinum electrode touching the cornea and a reference electrode in the pinna. A ground electrode was attached to the mouse's back. The flash stimulus was presented by a xenon strobe light placed 0.3 m in front of the mouse. Four consecutive responses were amplified and averaged using a MacLab/2e bioamplifier/data recorder running "Scope" software (ADInstruments, NSW, Australia). The interstimulus interval was increased from 30 sec (dimmest flash) to 5 min (brightest flash). Stimulus-response characteristics were generated by attenuating the maximum flash intensity (1.52 log cd s/m²) with neutral density filters over a range of 3 log units. After the final scotopic recording, the animals were light adapted for 10 min using a background light of 1.4 log cd/m² and photopic ERGs obtained. The a-wave amplitude was measured from the baseline to the trough of the a-wave response and the b-wave amplitude was measured from the trough of the a-wave to the peak of the b-wave. Data were expressed as the mean wave amplitude ± standard error of the mean (SEM; μVolts). Two-way repeated measures analysis of variance (ANOVA) was performed on log transformed data to compare the responses from the rAAV.RPE65-injected and uninjected Rpe65^-/- retinas. A post-hoc Bonferroni test was used to isolate significant differences (P < 0.05) between rAAV.RPE65-injected and uninjected Rpe65^-/- mice responses at each stimulus intensity. All mice were subjected to the same conditions for ERG measurements.

rAAV.RPE65-injected, age-matched uninjected Rpe65^-/- mice and age-matched control C57BL/6J mice were euthanased at various time points post-injection, and their eyes enucleated and fixed in 10% neutral buffered formalin for 2.5 hr. The eyes were then washed in PBS before being placed in 70% ethanol and embedded in paraffin, with care being taken to orientate the eyes so that the injection site was at a known and consistent location. Serial sections (5 μm) were cut on a Reichert-Jung 2040 microtome (Leica Microsytems, Australia), mounted on silanated glass slides, deparaffinized and rehydrated. All analyses that were performed using these eyes were carried out on sections from the region corresponding to the injection site.

For histological analysis and quantification, the sections were stained with hematoxylin and eosin, and the number of photoreceptor cells counted. Average cell numbers for each retina were established by counting the number of cells in a 100 μm section of the outer nuclear layer (using an eyepiece graticule and viewing the stained section with a 100X oil-immersion lens). Digital images of the outer nuclear layer of each section were recorded. Between three and five 100 μm regions within the subretinal bleb from each section were selected for counting. Care was taken to avoid the outer quarter to third of the retina where the retinal layers became thinner. Counts were made every 30–50 sections (150–250 μm) such that 15–20 counts were made per eye. The mean of these counts was then calculated to give an average number of photoreceptors per 100 μm for each eye. The counting was performed by 3 independent observers who were not given the identity of the samples.

Serial sections from rAAV.RPE65-injected and uninjected Rpe65^-/- eyes were rehydrated through graded alcohols, and then bleached by incubation in 0.25% potassium permanganate for 20 min followed by 1% oxalic acid for 5 min 28. The sections were rinsed several times in Tris buffer (50 mM, pH 7.2) containing 1% NaCl, then blocked in 10% normal goat serum for 1 hr. The sections were incubated at 4°C overnight with a rabbit anti-RPE65 antibody 25, rinsed three times in Tris buffer and then incubated for 2 hr at room temperature with alkaline phosphatase-conjugated, goat anti-rabbit IgG (1:100, Gibco Invitrogen, CA, USA). Immunodetection was carried out using SIGMA FAST Red TR/Naphthol AS-MX (Sigma Chemical Co., MO, USA) chromogen for 10–15 min, resulting in the formation of a red/pink precipitate. The sections were counterstained lightly with Meyer's hematoxylin and mounted in an aqueous mounting medium for analysis.

For flatmount immunohistochemistry, eyes were enucleated and the injection site was marked with indelible ink and then fixed whole for 30 min in 4% paraformaldehyde. The anterior segment of each eye was removed and the neuroretina separated from the sclera-choroid-RPE layers. The separated layers were placed in separate wells of a 96-well plate and blocked with 10% normal rabbit serum at room temperature for 1 hr. The layers were then incubated overnight at 4°C with primary antibodies, rabbit anti-RPE65 antibody and rabbit anti-short wavelength cone (SWC) opsin antibody, washed with Tris-buffered saline (TBS) and incubated for 2 hr at 4°C with goat anti-rabbit IgG conjugated with FITC (fluorescein isothiocyanate; Sigma Chemical Co.). After 3 washes in TBS, radial cuts were made to the neuroretina and the sclera-choroid-RPE layers which were mounted separately on slides with GVA mounting solution (Zymed, CA, USA) and coverslipped prior to examination. Areas within the injection subretinal bleb in rAAV.RPE65-injected Rpe65^-/- mice or the equivalent location in C57BL/6J and uninjected Rpe65^-/- mice were examined by fluorescence microscopy. The number of SWC opsin-positive photoreceptors was counted in five 100 μm² areas within the subretinal bleb and the results analyzed and graphed.

rAAV.RPE65-injected (n = 2), age-matched uninjected (n = 2) Rpe65^-/-, and age-matched C57BL/6J control mice were euthanased at 7 mo post-injection (8 mo of age) and their eyes enucleated, processed and sectioned as described previously. An Apoptosis detection assay was performed on these sections using the Dead End™ Colorimetric TUNEL Systems (Promega Corp.). The assay was performed as described in the manufacturer's instructions. When complete, the sections were counterstained with 0.5% methyl green for 10 min, briefly washed in water then 1-butanol, dehydrated with xylene, and mounted with DePeX mounting medium (BDH Laboratory Supplies, England, UK). Images of the outer nuclear layer were captured with an Olympus DP-7 digital camera (Olympus, NY, USA) mounted on a light microscope (Olympus BX60) using a 100X oil immersion lens. The relative level of apoptosis was then determined by expressing the number of TUNEL-positive nuclei as a percentage of the total nuclei over a 60 μm region of the retina. Three to five 60 μm regions were counted from each section, depending on the size of section, with care being taken to avoid the outer, thinning quarter of the retinas.

rAAV.RPE65-injected and uninjected eyes from Rpe65^-/- mice at 20 mo post-injection (21 mo of age) were first perfused with fixative (2.5% glutaraldehyde in cacodylate buffer, pH 7.4), then enucleated and fixed for a further 24 hr in fixative at 4°C. Following careful removal of the cornea and lens, the tissues covering the injection site and outside the injection site were trimmed into 1 mm³ blocks and re-immersed into fresh fixative for a further 24 hr at 4°C. After post-fixing in 1% osmium tetroxide, the tissues were processed for transmission electron microscopy (TEM) by conventional methods and embedded in Araldite. Semi-thin sections (1 μm) were stained with 0.5% toluidine blue in 5% borax and examined with a light microscope. After selecting the areas of interest, the blocks were trimmed under a dissecting microscope. Ultra-thin sections (70 nm) were then prepared on an ultramicrotome (LKB Nova, Sweden), stained with Reynolds lead citrate and examined in a Philips 410LS Transmission Electron Microscope at an accelerating voltage of 80 kV.

The presence of RPE65 expression following subretinal injection of rAAV.RPE65 and rAAV.RPE65.1 was monitored by immunohistochemistry using a rabbit anti-RPE65 antibody. The efficiency and specificity of the RPE65 antibody was confirmed using retinal sections from C57BL/6J and uninjected Rpe65^-/- mice. RPE65 immunoreactivity was readily detectable in the cytoplasm of RPE cells in C57BL/6J mice (data not shown), but was completely absent from those of uninjected Rpe65^-/- mice (Fig. 1B). No RPE65 immunoreactivity was seen in the photoreceptor layers of either C57BL/6J or uninjected Rpe65^-/- mouse retinas.

In a preliminary study, Rpe65^-/- mice were injected with either rAAV.RPE65 or rAAV.RPE65.1. Analysis of RPE/choroid and retina flatmounts of rAAV.RPE65-injected Rpe65^-/- mice showed that the area of RPE65 expression covered approximately 30% of the surface of the RPE/choroid flatmounts (corresponding to the size of the bleb created) with no RPE65 expression present in the flatmounted neuroretina. The RPE65 expression appeared contiguous within the injection area, although some small, scattered areas with no signal were seen (data not shown). In contrast, in rAAV-RPE65.1-injected Rpe65^-/- mice, RPE65 expression was not only present in the RPE/choroid flatmounts (again in an area of approximately 30% of the retina), but was also present in the neuroretina flatmounts where more RPE65-immunostained cells were detected. The RPE65 expression in both the RPE/choroids and neuroretina flatmounts of the rAAV-RPE65.1 injected eyes was weaker, and appeared more dispersed, probably due to the fewer number of cells transduced when compared to rAAV.RPE65-injected RPE/choroids flatmounts (data not shown). On the basis that rAAV.RPE65 was more efficient in transducing RPE cells,, and in order to target transduction of RPE cells only, subsequent studies were conducted using rAAV.RPE65.

A histological analysis of RPE65 immunoreactivity in the rAAV.RPE65-injected mice over time demonstrated that strong RPE65 positive RPE cells were visible from the injection site to up to 300–600 μm away, but still within the bleb created, at 1–2 mo (data not shown), 7 mo (Fig. 1A), 11 mo (Fig. 1C and 1D) and 18 mo (Fig. 1E and 1F) post-injection. However, the extent of RPE65 expression appeared to decrease at the latest time point. No RPE65 immunoreactivity was seen in either uninjected, age-matched control Rpe65^-/- mice, or Rpe65^-/- mice injected with the control rAAV.GFP construct (data not shown). There was no evidence of infiltrating immune cells in any of the eyes examined.

ERG analysis of Rpe65^-/- mice showed an improvement in the response of rAAV.RPE65-injected animals compared with uninjected, age-matched controls. A comparison of scotopic and photopic ERG responses from injected and uninjected mice is presented in Fig. 2. At 1–2 mo post-rAAV.RPE65 injection, an increase in the ERG b-wave amplitude was apparent (Fig. 2A and 2B, upper trace). A two-way repeated measures ANOVA of the stimulus-response characteristics (Fig. 3) demonstrated a significant (P < 0.001) difference in the scotopic b-wave amplitude between the control and rAAV.RPE65-injected mice. Post-hoc Bonferroni tests revealed a significant (P < 0.005) increase of the scotopic b-wave at all flash intensities above -0.9 log neutral density units (Fig. 3B). There was also a significant interaction between stimulus intensity and rAAV.RPE65-injection in the photopic b-wave amplitude (P < 0.05). The post-hoc Bonferroni tests also revealed a significant (P < 0.05) increase of the photopic b-wave at the brightest flash intensities (Fig. 3B). No statistically significant improvement in a-wave amplitude was seen at this time point (Fig. 3A, P > 0.05). At 7 mo and 11 mo post-injection, no differences were found in the ERG a-wave (Fig. 2B and 2C) or b-wave amplitudes (Fig. 2B,2C, 3C and 3D) recorded from rAAV.RPE65-injected mouse eyes when compared with responses recorded from uninjected, age-matched controls under either scotopic or photopic conditions. Additional rAAV.GFP-injected Rpe65^-/- control mice showed ERG signals equivalent to those of uninjected controls (data not shown).

Histological analysis of the retinas of uninjected Rpe65^-/- mice showed a slow, progressive degeneration of photoreceptors. In brief, at the early age of 1–2 mo, the retinas of uninjected Rpe65^-/- mice appeared normal, except for the less organized appearance of the photoreceptor outer segments (Fig. 4A). The outer nuclear layer of uninjected Rpe65^-/- mice aged 6–12 mo were visibly thinner and the outer segments appeared highly disorganized when compared to age-matched C57BL/6J controls. At 12 mo and older (Fig. 4B), the difference in the outer nuclear layer thickness was very significant when compared to age-matched C57BL/6J mice (Fig. 4C) and by 21 mo of age, the outer nuclear layer was completely absent (data not shown). The morphologic difference was quantified by counting the number of photoreceptor nuclei in the eyes of uninjected Rpe65^-/- mice at 2, 5, 7, 11, 17 and 24 mo post-injection and comparing them to those of age-matched C57BL/6J mice. A statistically significant decrease (P < 0.05, Student's t-test) in photoreceptor number was obtained for uninjected Rpe65^-/- mice older than 3 mo (Fig. 4D), reflecting the progressive loss of photoreceptor cells in these mice. Subsequent comparison of rAAV.RPE65-injected with age-matched, uninjected control Rpe65^-/- mice indicated that no statistically significant difference in the number of photoreceptors around the injection site was seen at any of the time points (Fig. 4D; P > 0.05, Student's t-test), suggesting that there was no photoreceptor rescue or slow down in photoreceptor loss following rAAV.RPE65 injection. The lack of photoreceptor rescue was reflected by the lack of difference in the number of apoptotic cells in rAAV.RPE65-injected eyes of Rpe65^-/- mice (Fig. 5A) when compared to the contralateral uninjected eyes (Fig. 5B). At 7 mo post-injection (8 mo of age), the number of apoptotic cells in both the uninjected and rAAV.RPE65-injected Rpe65^-/- appeared higher than those in age-matched C57BL/6J controls (Fig. 5C). Analysis of the 60 μm regions of uninjected and rAAV.RPE65-injected Rpe65^-/- eyes at 7 mo post injection (8 mo of age) showed that 5.8 ± 1.9% and 2.7 ± 1.7%, respectively, of the remaining photoreceptors were apoptotic (P > 0.01, Student's t-test, Fig. 5D).

Electron microscopy of rAAV.RPE65-injected and uninjected Rpe65^-/- mouse eyes at 20 mo post injection (21 mo of age) revealed the presence of retinyl ester lipid droplets that are characteristic of Rpe65^-/- mice 7. However, a direct comparison of the RPE in the rAAV.RPE65-injected mice (Fig. 6A) with the uninjected, age-matched control (Fig. 6B) showed a striking difference between the amounts of lipid inclusions present in these eyes. In contrast, electron microscopy of sections taken from outside the subretinal bleb of rAAV.RPE65-injected eyes showed no difference between the numbers of lipid inclusions when compared to sections from uninjected eyes (data not shown). In addition to the reduction in numbers of lipid droplets, the layer of basal infoldings was also thinner in rAAV.RPE65-injected eyes (Fig. 6A and 6B).

Immunostaining using the anti-SWC opsin antibody demonstrated the presence of SWC opsin-positive cells scattered throughout the flatmounted neuroretinas of 8 month old C57BL/6J mice (Fig. 7A). The number of SWC opsin-positive cells was significantly lower in uninjected Rpe65^-/- mouse retinas, with only a small number of SWC opsin-positive cells being seen in the neuroretinas of either 3 week (Fig. 7B) or 3 month old Rpe65^-/- mice (data not shown). By 8 months of age no SWC opsin-positive cells were visible in the neuroretinas of uninjected Rpe65^-/- mice (Fig. 7C). Examination of flatmounted neuroretinas of 8-month-old rAAV.RPE65-injected Rpe65^-/- mice revealed the presence of numerous SWC opsin-positive cells in an area coinciding with the subretinal bleb and, at a higher density, around the injection site (Fig. 7D). Counting and analysis of the number of SWC opsin-positive cells in the C57BL/6J control (n = 5), uninjected Rpe65^-/- mice (n = 5) and rAAV.RPE65-injected Rpe65^-/- eyes (n = 5) showed that the reappearance of the SWC opsin-positive cells in rAAV.RPE65-injected Rpe65^-/- mice was significant, reaching up to 50% of that seen in age-matched C57BL/6J mice (Fig. 7E).