Development of an optimized AAV2/5 gene therapy vector for Leber congenital amaurosis owing to defects in RPE65

Leber congenital amaurosis is a group of inherited retinal dystrophies that cause severe sight impairment in childhood; RPE65-deficiency causes impaired rod photoreceptor function from birth and progressive impairment of cone photoreceptor function associated with retinal degeneration. In animal models of RPE65 deficiency, subretinal injection of recombinant adeno-associated virus (AAV) 2/2 vectors carrying RPE65 cDNA improves rod photoreceptor function, and intervention at an early stage of disease provides sustained benefit by protecting cone photoreceptors against retinal degeneration. In affected humans, administration of these vectors has resulted to date in relatively modest improvements in photoreceptor function, even when retinal degeneration is comparatively mild, and the duration of benefit is limited by progressive retinal degeneration. We conclude that the demand for RPE65 in humans is not fully met by current vectors, and predict that a more powerful vector will provide more durable benefit. With this aim we have modified the original AAV2/2 vector to generate AAV2/5-OPTIRPE65. The new configuration consists of an AAV vector serotype 5 carrying an optimized hRPE65 promoter and a codon-optimized hRPE65 gene. In mice, AAV2/5-OPTIRPE65 is at least 300-fold more potent than our original AAV2/2 vector.


Virus preparation and titre
Recombinant vectors were produced through a triple transient transfection method. The plasmid construct, AAV serotype-specific packaging plasmid and helper plasmid, in a ratio of 1:1:3 at 20 mg total DNA per ml of DMEM, were mixed with Polyethylenimine (Polysciences Inc.) to a final concentration of 50 mg.mL -1 and incubated for 10 min at room temperature to form transfection complexes that were added to 293T cells at 50 µg DNA per 15-cm plate and left for 72 h. The cells were collected, concentrated and lysed by freeze-thaw (3x) in PBS to release the vector. Recombinant AAV was bound to an AVB Sepharose column (GE Healthcare), and eluted with 50mM Glycine pH2.7 into 1M Tris pH 8.8. Vectors were washed in 1x PBS and concentrated to a volume of 100-150 µl using Vivaspin 4 (10 kDa) concentrators. Viral particle titres were determined by quantitative real-time PCR (qPCR) using an ITR binding assay as previously described (Aurnhammer et al, 2012).

Subretinal injections
Mice were anesthetised with a single intraperitoneal injection of a 0.01-ml/g mixture of Domitor (1 mg/ml medetomidine hydrochloride), ketamine (100 mg/ml), and water at a ratio of 5:3:42 before surgical procedures. Rabbits were anesthetised with a single intraperitoneal injection of a 0.5-ml/kg mixture of Domitor (1 mg/ml medetomidine hydrochloride) and 0.25ml/kg ketamine (100 mg/ml). Subretinal injections were performed under direct retinoscopy thorough an operating microscope. The tip of a 1.5-cm, 34-gauge hypodermic needle (Hamilton) was inserted tangentially through the sclera of the mouse eye, causing a self-sealing wound tunnel. A 30-gauge needle was used for injections into rabbit eyes. The needle tip was brought into focus between the retina and retinal pigment. Mice received double injections of 2 µl each to produce bullous retinal detachments in the superior and inferior hemisphere around the injection sites. Rabbits received one injection of 200 µl in the superior hemisphere. Eyes were assigned as treated and (contralateral) control eyes using randomisation software.

Western Blot of cellular protein extracts
Protein extracts were quantified in a Bio-Rad protein assay (Bio-Rad, Hemel Hempstead, UK). Equal amounts of each sample were loaded onto 6% polyacrylamide gels and, after gel electrophoresis, transferred to nitrocellulose membrane for Western blotting. The membrane was cut in using the loading ladder as guide to separate parts of the membrane according to blotted proteins (RPE65 or Histone 2B). After blocking the membrane in block solution (5% skimmed milk powder-1% bovine serum albumin [BSA; Sigma-Aldrich]-0.05% Tween 20 in PBS) overnight at 4°C, mouse monoclonal anti-RPE65 antibody [diluted 1:1000; Ab13826, Abcam, Cambridge, UK] or mouse monoclonal anti-H2B antibody [diluted 1:5000; 2934S, New England Biolabs, Wilbury, UK] was added and left overnight at 4°C. Membrane was thoroughly washed in PBS-0.05% Tween 20 and incubated with a horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG antibody (diluted 1:5000; Dako, Cambridge, UK) in block solution for 1 hr at room temperature. After PBS-0.05% Tween 20 washes, signal was developed with Amersham enhanced chemiluminescence (ECL) Plus Western blotting detection reagents (GE Healthcare, Chalfont St Giles, UK).

Electroretinographic analysis
Electroretinograms (ERGs) were recorded from both eyes of Rpe65-/-mice, C57BL6/J wildtype mice and New Zealand White wild-type rabbits. All animals were dark adapted overnight before ERG recordings. Following anaesthesia, the pupils were dilated with a drop of Minims Tropicamide 1% (Bausch & Lomb/Chauvin Pharmaceuticals, Essex, UK). Midline subdermal ground and mouth reference electrodes were first placed, followed by eye electrodes that were allowed to lightly touch the corneas. A drop of Viscotears 0.2% liquid gel (Dr. Robert Winzer Pharma/OPD Laboratories, Watford, UK) was placed on top of the electrodes to keep the corneas moistened during recordings. ERGs were recorded with commercially available equipment (Espion E2; Diagnosys, Lowell, MA). Bandpass filter cutoff frequencies were 0.312 Hz (low-frequency cutoff) and 1000 Hz (high-frequency cutoff ). Scotopic, rod-mediated responses were obtained from dark-adapted animals at the following increasing light intensities: 0.001, 0.01, 0.1, 1 and 3 cds/m 2 . Ten responses per intensity were recorded for the first three intensities with 10-sec dark adaptation between each. Five responses were recorded for all the subsequent steps with 30-sec dark adaptation between each. Responses were averaged for each intensity. Photopic, cone-mediated responses were performed after a 10-min light adaptation on a background light intensity of 30 cd/m 2 , which was used as background intensity for the duration of photopic recordings, flash and flicker. Recordings were obtained at the following increasing light intensities: 0.1, 1, 3, 5 and 10cds/m 2 . Twenty-five responses were averaged for each intensity, with a 60-sec light adaptation interval between each step.

Histology
Animals were killed, the eyes enucleated and the cornea, lens and iris removed. For retinal sections, eyecups were fixed in 4% paraformaldehyde for 1 h and incubated in 20% sucrose for 1 h at room temperature, before embedding in optimal cutting temperature medium. Twelve µm cryosections were cut in sagittal orientation, counterstained with 4',6-diamidino-2-phenylindole (DAPI) and mounted with DAKO fluorescent mounting media (DAKO, S3023, Denmark). Images were acquired by confocal microscopy (Leica DM5500Q).