Forty dogs were studied. Twenty-eight dogs homozygous for the canine RPE65
mutation (), and 1 unaffected control (), were part of a research strain of mixed-breed dogs maintained at the Retinal Disease Studies Facility (Kennett Square, PA, USA). The disease in this strain derives from a single affected briard dog and is caused by a 4-bp deletion in the canine RPE65
gene, as described previously [13
]. Molecular diagnostic testing has determined that this strain is homozygous normal for other genes/loci responsible for inherited retinal degeneration in dogs (prcd, erd, CNGB3, PDE6B, Rho, RPGR
). Two purebred briard dogs (Exp1817, Exp2818; ), affected by the same RPE65
mutation as the colony dogs, were also studied as affected, untreated controls. In addition, 14 eyes from 9 normal control dogs were used for ERG studies. All procedures involving animals were undertaken in accordance with the guidelines of the U.S. Public Health Service’s policy on the humane care and use of laboratory animals. Twenty-nine eyes of 19 affected dogs each received a single subretinal injection containing a therapeutic vector (); 26 of these eyes (17 dogs) were subsequently tested at least once by ERG. Fourteen eyes of 11 affected dogs () received an intravitreal injection of vector, a sham injection, or no injection, and subsequently each of these eyes was also tested at least once by ERG. Eyes were harvested at selected time points posttherapy to evaluate the results of treatment by morphology, immunohistochemistry, retinoid analyses, and molecular studies (). Thirty-seven eyes from 22 dogs were harvested for morphological evaluation, including 21 for immunohistochemical examination (). These included 4 eyes from the 2 purebred affected briard dogs and 33 eyes from 20 colony dogs. Eighteen of these eyes had each received a subretinal vector injection, 7 had received an intravitreal injection, and 12 (including the 4 purebred briard eyes) had received no therapy.
Five types of therapeutic AAV vectors, identified as AAV2/2-CBA-cRPE65, AAV2/5-CBA-hRPE65, AAV2/2-CBA-hRPE65, AAV2/1-CBA-hRPE65, and AAV2/1-RPE08-hRPE65, were used (). All vectors are flanked by 143-bp AAV2 inverted terminal repeats and contain a 199-bp SV40 polyadenylation signal sequence. AAV2/2, AAV2/1, and AAV2/5 vectors are packaged into serotype 2, 1, or 5 capsids, respectively. CBA indicates the 1680-bp hybrid chicken β-actin promoter, encompassing a cytomegalovirus immediate early enhancer (381 bp), the proximal chicken β-actin promoter (283 bp), and the chicken β-actin intron 1 flanked by exon 1 and exon 2 sequences [29
]. RPE08 is an 823-bp human RPE65-specific promoter (−1 to −822). cRPE65 is a 1656-bp canine RPE65 cDNA, and hRPE65 is a 1602-bp human RPE65 cDNA. AAV2/1-CBA-hRPE65 also contains a 594-bp woodchuck hepatitis virus posttranscriptional element. All AAV vectors were produced and purified identically according to Zolotukhin et al
] but with modifications. HEK 293 cells (ATCC) were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 5% fetal bovine serum and antibiotics. A CaPO4
transfection precipitation was set up by mixing a 1:1 molar ratio of the rAAV vector plasmid and the helper plasmid pDG(4), which contained all the helper functions required for production of the vector, including the desired AAV serotype capsid gene. For serotype 2 vectors the capsid gene was from wild-type serotype 2 AAV; for serotype 1 vector a pseudotyped virus was produced with a helper plasmid containing an AAV serotype 1 capsid gene, and for the serotype 5 vector a pseudotyped virus was produced with helper plasmid containing an AAV serotype 5 capsid gene. For each vector the appropriate two-plasmid DNA precipitate was added to 1100 ml of DMEM and the mixture applied to 293 cell monolayers in a cell factory (Nalge Nunc International, Rochester, NY, USA). The transfection was allowed to incubate at 37°C for 60 h, after which the cells were harvested and lysed by three freeze/thaw cycles. The crude lysate was clarified by centrifugation and the resulting vector-containing supernatant divided between four discontinuous iodixanol step gradients and run at 350,000g
for 1 h. Five milliliters of each 60–40% step interface was removed, and the combined vector fractions were further purified and concentrated by column chromatography on a 5-ml HiTrap, Q Sepharose column using a Pharmacia ATKA FPLC system. The vector was loaded in 20 mM NaCl, pH 8.0, and eluted from the column using a 500 mM NaCl, pH 8.0, step gradient. The appropriate eluted fractions were then pooled and concentrated and the buffer was exchanged with PBS in a Biomax 100K concentrator (Millipore, Billerica, MA, USA). Vector purity was assessed by silver-stained SDS–polyacrylamide gel electrophoresis [30
]. Each vector was titered for physical particles by quantitative competitive PCR [32
] and AAV vectors were then stored at −80°C in PBS prior to use.
Surgical procedures and postsurgical treatment and evaluation
Five cubic centimeters of blood for baseline serology studies was collected by venipuncture prior to treatment, and then animals were anesthetized with thiopental/isoflurane. Subretinal injections were performed as described [7
] after injecting 5–10 cc sterile saline retro-orbitally to prevent rotation of the eye. Briefly, after mydriasis, an anterior chamber paracentesis was performed with a 30-gauge needle to provide space for the vector and to obtain fluid for baseline intraocular antibody measurements. A 30-gauge anterior chamber cannula (Storz) was inserted through a sclerotomy incision and gently pressed against the neural retina at the desired injection site. A dose volume between 100 and 200 μl was delivered subretinally, thereby creating a localized dome-shaped retinal detachment (“bleb”). The vector dose delivered ranged from approximately 1010
particles. The retinal location of the bleb was observed at the time of surgery and documented by indirect ophthalmoscopy, fundus drawings, and photography. Retinal vessels were assessed to ensure that they remained well perfused during and following the procedure. Four milligrams of Kenalog (triamcinolone acetonide; 40 mg/ml; Bristol–Meyers–Squibb, New York, NY, USA) was injected subconjunctivally, and PredG ointment (Allergan Pharmaceuticals, Irvine, CA, USA) was applied to the corneas while the animals recovered from anesthesia. Following surgery, the dogs were monitored by routine clinical ocular examinations using binocular indirect ophthalmoscopy and biomicroscopy after mydriasis (1% tropicamide); flattening of the subretinal bleb or presence of ocular inflammation was recorded. Immediately following surgery, retinal location and extent of the area of subretinal treatment injection, apparent as a bleb at the time of the surgery, was documented with indirect ophthalmoscopy and fundus drawings. The area of the bleb was quantified from the drawings and specified as a (unitless) fraction of the area of a standard tapetal zone. The location of the injection area was specified as superior (tapetal) or inferior (nontapetal) in most cases; in some eyes, however, the injection area was on the boundary between tapetal and nontapetal zones and was specified as such.
Dogs were dark-adapted overnight and anesthetized as described [7
]. Pupils were dilated (cyclopentolate, 1%; phenylephrine,10%), and pulse rate, oxygen saturation, and temperature were monitored. Full-field ERGs were recorded with Burian–Allen contact lens electrodes and a computer-based system. Low-energy (10 μs duration; maximum luminance of unattenuated white flash 0.4 log scot-cd s m−2
) and high-energy (1 ms duration; maximum luminance of unattenuated white flash 3.7 log scot-cd s m−2
) flashes were used under dark-adapted and light-adapted (1.5 log cd m−2
at 1-Hz stimulation, 0.8 log cd m−2
at 29-Hz stimulation) conditions [7
Dark-adapted dogs were euthanized and enucleated (all procedures performed under dim red light). Retina, separated from RPE, was divided into six sectors, three from the superior tapetal zone and three from the inferior nontapetal zone. Tissue samples were double wrapped in aluminum foil and stored at −80°C until use. All experimental procedures related to the analysis, derivatization, and separation of retinoids were undertaken as described previously for mouse eyes [8
] with minor modifications as described below. Prior to extraction, retinals were derivatized to oximes with hydroxylamine for better separation under our chromatographic conditions. Retinoid analysis was performed on an Agilent 1100 series high-pressure liquid chromatograph (HPLC) equipped with a diode array detector and Agilent Chemstation A.10.01 software. A normal-phase column (Beckman Ultrasphere Si 5μ, 4.6 × 250 mm) and an isocratic solvent system of 0.5% ethyl acetate in hexane (v/v) for 15 min followed by 4% ethyl acetate in hexane for 65 min at a flow rate of 1.4 ml/min at 20°C (total 80 min) with detection at 325 nm were used.
Histopathology and immunocytochemistry
Thirty-seven eyes were studied morphologically from 20 affected dogs, purposely bred for these studies, and 2 affected purebred briard dogs (4 and 10.5 months of age). These included 18 eyes receiving subretinal injections, 7 receiving intravitreal injections, and 12 receiving no therapy (). Posttreatment intervals ranged from 3.5 months to 2 years. Retinal sections for morphologic studies were prepared using a triple fixation protocol [33
] prior to embedding in plastic, a 4% paraformaldehyde fixation for OCT embedding and immunocytochemistry [34
], or Bouin’s solution followed by processing for standard paraffin embedding and sectioning for histopathological examination and immunochemistry. For treated eyes, plastic- or OCT-embedded tissues were oriented such that the sections extended through the center of the treated area; untreated areas from adjoining and other quadrants were also included for analysis. For immunocytochemical studies, sections from OCT-embedded retinas were labeled with rabbit anti-mouse RPE65 polyclonal antibody (PETLET; gift from Dr. Michael Redmond), monoclonal antibody K16-107C directed at the C-terminal domain of opsin [35
], DAPI, and/or PNA to label the insoluble extracellular domain surrounding the cones [36
]. Secondary antibodies included goat anti-rabbit IgG conjugated to either Alexa Fluor 488 (green) or Alexa Fluor 568 (red) and goat anti-mouse IgG conjugated to Alexa Fluor 568 (Molecular Probes, Eugene, OR, USA). Sections were examined with a Zeiss Axioplan microscope using epifluorescence and DIC optics. Images were digitally captured (Spot 3.3; Diagnostic Instrument, Inc., Sterling Height, MI, USA) and imported into Adobe Photo-Shop (Adobe Systems, San Jose, CA, USA).
Samples for serology, virology, and extraocular transgene expression
Serum samples were obtained from all dogs receiving therapy, immediately prior to surgery, by venipuncture 3 weeks after injection and, again, terminally. Conjunctival swabs for viral isolation were taken at multiple time points following surgery. Following euthanasia with a pentobarbital overdose, eyes that were enucleated for other studies had fluid samples collected from the anterior chamber and vitreous. All samples were stored at −80°C until used for immunology studies.
Immunology: ELISA to determine Anti-RPE65 antibody response after AAV-RPE65 gene therapy
Antigen was prepared from human RPE65 cDNA amplified from plasmid pAAV2.1CMV-hRPE65  using forward primer 5′-AGGAATTCCATGTCTATCCAGGTTGAGCATC-3′ and reverse primer 5′-CAGAATTCTCAAGATTTTTTGAACAGTCCATG-3′. The 1.6-kb product was digested with EcoRI and subcloned into pGEX3X (Pfizer, New York, NY, USA), creating an in-frame fusion of hRPE65 with GST. Both GST-hRPE65 and GST (control) proteins were expressed in BL21 Codon-Plus RIL bacteria (Stratagene, La Jolla, CA, USA). Five milliliters of an overnight culture was used to inoculate 200 ml LB broth; bacterial pellets were washed with PBS and resuspended in 20 ml of lysis buffer (5% lithium dodecyl sulfate, 10 mM Tris, pH 8.2) supplemented with protease inhibitors. DNA was removed by passing the lysates over a column of 425–600 μm-sized, acid-washed glass beads (Sigma Chemical Co., St. Louis, MO, USA). Cleared lysates were aliquoted and stored at −80°C. To verify hRPE65 expression, each bacterial lysate was run on a 10% Bis-Tris NuPAGE gel (Invitrogen, Carlsbad, CA, USA) with Mops running buffer and then electroblotted onto Hybond ECL (Amersham, Piscataway, NJ, USA). The blot was probed with 1:1000 diluted rabbit anti-RPE65 antiserum (PETLET), followed by 1:2000 diluted, HRP-conjugated, donkey anti-rabbit immunoglobulin (Amersham) and finally ECL Plus detection reagent (Amersham). A band of approximately 90 kDa was detected in lanes containing GST-RPE65 lysates and was absent in lanes containing only GST lysates. The ELISA validation assay demonstrated that the reaction conditions tested specifically for antibodies to RPE65.
ELISA validation assay
One hundred microliters of antigen (1:100 dilution in 0.1 M sodium bicarbonate, pH 9.6) was aliquoted per well of a 96-well high-binding EIA/RIA plate (Corning, Corning, NY, USA) and incubated overnight at 4°C. After coating with antigen, the wells were blocked with 1% BSA and sequentially incubated with 1:2000 diluted rabbit anti-RPE65 antiserum (PETLET) or control rabbit anti-β-galactosidase antiserum (Chemicon, Temecula, CA, USA), followed by 1:2000 diluted, HRP-conjugated, donkey anti-rabbit immunoglobulin (Amersham) and p-phenylenediamine dihydrochloride substrate (Sigma–Aldrich Corp., St. Louis, MO, USA). Between steps, the wells were washed with 0.05% Tween 20 in PBS. The OD490 was determined after quenching with H2SO4 using a plate reader (Wallac Victor, Perkin–Elmer, Boston, MA, USA).
Analysis of sera, anterior chamber fluid, and vitreous of treated dogs
Samples from all animals were screened for immunoreactivity to RPE65 protein. Serum from an untreated 3-week-old unimmunized puppy was used as a negative control. All samples were stored at −80°C from the time of collection until their use. The GST and GST-RPE65 test antigen preparations as well as LCI-GP parvo/distemper vaccine (positive control; Fort Dodge Animal Health Division of Wyeth) were diluted 1:100 in 0.1 M sodium bicarbonate, pH 9.6, and 100 Al of diluted antigen was aliquoted per well of a 96-well high-binding EIA/RIA plate (Corning) and incubated overnight at 4°C. After being coated with antigen, the wells were blocked with 1% BSA and sequentially incubated with 1:50 diluted specimen and subsequently analyzed as above.
To confirm the results obtained by ELISA, sera from positive (AAV-treated; BR29) and negative (untreated and unimmunized puppy) control dogs were used as probes for Western analysis. Results were compared with a blot probed with rabbit anti-RPE65 antiserum. One microliter of each antigen was run in triplicate on a 10% Bis-Tris NuPAGE gel (Invitrogen) with Mops running buffer and then electroblotted onto Hybond ECL (Amersham). The blots were probed with 1:200 diluted dog serum or 1:1000 diluted rabbit anti-RPE65 antiserum; followed by 1:2000 diluted, HRP-conjugated, sheep anti-dog immunoglobulin (Sigma) or donkey anti-rabbit immunoglobulin (Amersham) and ECL Plus detection reagent (Amersham).
Immunology: antibodies to AAV2/2
Samples were analyzed for anti-bodies to AAV type 2 capsid proteins. Enhanced protein binding ELISA plates (Costar, Corning, NY, USA) were coated for 2 h at room temperature with antigen using 1.8 × 109 particles/well of AAV2/2 in bicarbonate buffer, pH 9.6. Plates were then washed, blocked, and incubated with diluted (1:20, 1:100) serum and intraocular fluid. Saline and serum from an uninjected, unimmunized puppy were used as negative controls. Samples were applied to wells in triplicate and were incubated overnight at 4°C. Human serum containing high levels of anti-AAV2/2 antibodies served as a positive control. Samples were then washed and incubated for 2 h at room temperature with a 1:1000 dilution of alkaline phosphatase-conjugated rabbit anti-dog IgG (Sigma, 100 Al/well). After washing, color was developed using Sigma Fast paranitrophenyl phosphate substrate (Sigma). The plates were read at an optical density at 405 nm.
Molecular analyses of extraocular transgene expression
Sera, samples from conjunctival swabs, and other frozen organ tissues were analyzed for the presence of RPE65 transcript, by RT-PCR, or transgene, by PCR. For RT-PCR studies of frozen extraocular tissues, RNA was extracted from 5–10 mg of tissue using the RNeasy Mini Kit (Qiagen, Inc., Valencia, CA, USA), and RNA (10 ng) was subsequently reverse transcribed and PCR was performed to amplify a segment of the canine RPE65 cDNA. Two sets of primers were used in two different reactions, using the GeneAmp RNA PCR Kit (Applied Biosystems, Foster City, CA, USA). An RT-PCR assay was designed to discriminate wild-type RPE65 transcript from that containing the briard mutation. Forward primer 5′-CATAACGGAATTTGGCACCT-3′ (JB7) and reverse primer 5′-CAGGGGAATTGTACGACGAC-3′ (JB8) amplify a 219-bp product from canine cDNA. The forward primer overlaps the briard deletion and amplifies only wild-type RPE65 when the primer is annealed at 10–12°C above its Tm. A second set of primers (5′-CATAACGGAATTTGGCACCT-3′ (JB5) and 5′-CAGGGGAATTGTACGACGAC-3′ (JB6)) flanks the region containing the briard deletion and amplifies a 396-bp product from both wild-type and mutant RPE65 under the same PCR conditions. PCR was carried out for 40 cycles with annealing at 51°C for 30 s and extension at 72°C for 1 min per cycle with a final extension of 10 min at 72°C. The PCR products were resolved on a 2% agarose gel. Additional PCRs designed to amplify the wild-type AAV REP DNA sequence were performed using primers forward 5′-TCCTTCAATGCGGCCTC-3′ and reverse 5′-TCATCTTCCCCTCCTCC-3′. PCR was carried out for 36 cycles with annealing at 57°C for 1 min and extension at 72°C for 1 min per cycle with a final extension of 10 min at 72°C.