Evaluation of AAV-DJ vector for retinal gene therapy

Animals

All animal experiments were conducted in accordance with protocols approved by Institutional Animal Care and Use Committee of Keio University School of Medicine (#2808). Only adult C57BL/6 mice were used in this study. All animals were maintained under a 12∕12-h light/dark environment.

Vector production and purification

pAAV-CAGGS-EGFP-WPRE was used. The CAGGS promoter contains a cytomegalovirus enhancer and a chicken β-actin promoter (Niwa, Yamamura & Miyazaki, 1991). All serotypes of AAV vectors were prepared using AAV Helper Free Packaging System (Cell Biolabs, San Diego, CA, USA). The serotypes were produced in HEK 293 cells using a helper virus-free system and purified using two CsCl₂ density gradients and titrated by quantitative polymerase chain reaction. Final preparations were dialyzed against phosphate-buffered saline (PBS) and stored at −80 °C.

Virus injection

The mice were anesthetized with pentobarbital sodium at the dose of 50 mg/kg body weight and placed on a heating pad that maintained their body temperature at 35–36 °C throughout the experiments. An aperture was made next to the limbus through the sclera with a 30-gauge disposable needle, and a 33-gauge unbeveled blunt-tip needle on a Hamilton syringe was introduced through the scleral opening into the vitreous space for intravitreal injections and introduced through the scleral opening along the scleral interior wall into the subretinal space for subretinal injections. Each eye received 1 µl of vehicle (PBS) or vector at a titer of 1.4 × 10e12 vg/ml.

Fundus photography

The mice were anesthetized with pentobarbital sodium at the dose of 50 mg/kg body weight and the pupils were dilated with a mixed solution of 0.5% tropicamide and 0.5% phenylephrine (Mydrin-P; Santen, Osaka, Japan). Live fundus images after the AAV injection were captured using a stereomicroscopy (M165-FC; Leica Microsystems, Wetzlar, Germany) with a glass slide and water to cancel the refraction. Fluorescence intensity was measured from photographs taken under the same conditions using ImageJ software (National Institutes of Health, Bethesda, MD, USA).

Preparation of retinal flat mounts and cryosections

Two months after vector injection, the mice were humanely euthanized. The eyes were removed and fixed with 4% paraformaldehyde in PBS for 1 h, and the cornea and lens were removed. To make flat mounts, the entire retina was dissected carefully from the eyecup and radial cuts were made from the edges to the retinal equator. For cryosections, the eyecups were washed in PBS followed by immersion in 30% sucrose in the same buffer overnight. The eyecups then were cryoprotected in O.C.T. Compound (Sakura Finetek Japan, Tokyo, Japan) and cryosectioned into 12-µm-thick transverse retinal sections.

Immunohistochemistry

Transduced mouse retinas were dissected and fixed in 4% paraformaldehyde for 30 min at room temperature. The retinas were incubated in PBS with 1% Triton X-100, 0.5% Tween 20 for 1 h at room temperature and in 10% normal goat serum for 1 h at room temperature and then incubated overnight at 4 °C with primary antibody: Brn3a (1:100; Santa Cruz Biotechnology, Santa Cruz, CA, USA), rhodopsin (1:500; Abcam, Cambridge, UK), PKCα (1:500; Abcam), calbindin (1:500; Abcam) and glutamine synthetase (GS) (1:500; Abcam) in blocking buffer. Secondary anti-rabbit IgG and anti-mouse IgG conjugated with Alexa TM594 and Alexa TM555, respectively (1:1,000; Molecular Probes), were applied for 1 h at room temperature. The retinas then were flat-mounted and the sections mounted on slide glass.

Fluorescent imaging

The retinal flat-mount images were obtained using a fluorescence microscope (BZ-9000, Keyence, Osaka, Japan). Fluorescent images of the cryosections were obtained using a confocal microscope (LSM710, Carl Zeiss, Jena, Germany).

ERG analyses

The scotopic ERGs were recorded according to a previous report (Zheng et al., 2012). The animals were dark-adapted for 12 h and prepared under dim red illumination. Damaged control mice with light-induced retinopathy were prepared according to a previous report (Kubota et al., 2010). The mice were anesthetized with a combination of midazolam, medetomidine, and butorphanol tartrate at the doses of 4, 0.75, and 5 mg/kg body weight and placed on a heating pad that maintained their body temperature at 35–36 °C throughout the experiments. The pupils were dilated with a mixed solution of 0.5% tropicamide and 0.5% phenylephrine (Mydrin-P, Santen, Osaka, Japan). The ground and references electrodes were placed subcutaneously into the tail root and between the eyes, respectively. The active contact lens electrodes (Mayo, Inazawa, Japan) were placed on the cornea. Recordings were performed using the PuRec system (Mayo) and filtered through a bandpass filter ranging from 1 to 100 Hz to yield the a- and b-waves. Light pulses of 2.0 log cd-s/m2 were delivered via a full-field light stimulator LS-100 (Mayo).

Statistical analysis

Statistical analysis was performed using SPSS software version 25 for Windows (IBM, Armonk, NY, USA). The criterion for statistical significance was p < 0.05, and the unpaired t-test or analysis of variance was performed.

Time course of enhanced green fluorescent protein (EGFP) expression in murine retina after AAV intravitreal injection

The purified AAV-2-CAGGS-EGFP or AAV-DJ-CAGGS-EGFP virus vector (hereafter referred to as AAV-2 or AAV-DJ) at a titer of 1.4 × 10e12 vg/ml was injected intravitreally and the changes over time in the EGFP signals in the retinas of live mice were observed using a fluorescence stereoscopic microscope. A fluorescent signal was confirmed 1 week after the injection and peaked at 8 weeks after the injection in both serotypes (Fig. 1A). AAV-DJ showed a rapid rise in fluorescence intensity, and its fluorescence intensity (n = 6 retinas) was significantly higher at 1 week after injection than that of AAV-2 (n = 6 retinas; p < 0.001, unpaired t-test) (Fig. 1B). Thereafter, the fluorescence intensity of AAV-2 (n = 6 retinas) also increased and tended to be higher than AAV-DJ at 4 weeks (n = 6 retinas; p = 0.11, unpaired t test) (Fig. 1C). We then evaluated the strength and efficacy of gene expression in retinal ganglion cells (RGCs) by investigating localization of expression by co-labeling with Brn3a, a reliable marker to identify and quantify RGCs (Quina et al., 2005). Confocal microscopy images of retinal flat mounts showed strong EGFP expression in RGCs and this expression was co-localized with the Brn3a labeling (Figs. 1D, 1E). Cell quantification in such images indicated that 81 ± 12.2% of these Brn3a-positive RGCs also expressed EGFP in the AAV-2 injected retinas (n = 24 images, n = 6 retinas) compared with 67 ± 72.6% in the AAV-DJ injected retinas (n = 24 images, n = 6 retinas; p = 0.095, unpaired t-test) (Fig. 1F).

Histologic comparison of EGFP expression mediated by AAV-2 vs. AAV-DJ

Two months after the intravitreal injection, EGFP expression was observed across the ganglion cell layer (GCL) side of the retinal flat mounts with both serotypes (Figs. 2A, 2C). Uniform EGFP expression was observed in the retinas injected with AAV-2 (Fig. 2A). In contrast, accumulation of EGFP signaling was observed with AAV-DJ injection in particular areas such as around the blood vessels covered with Mueller glia’s protrusions (Figs. 2E, 2F, Fig. S1). Cross-sections and immunohistochemical analysis showed EGFP fluorescence in RGC cell soma and axons, amacrine cells and horizontal cells in both serotypes (Figs. 2B, 2G, Fig. S2), whereas expression in Mueller cells (Figs. 2D, 2J–2L), bipolar cells, and rod photoreceptors (Figs. 3A–3C) was only found with AAV-DJ.

However, EGFP expression was detectable only around the injection site after subretinal injection of both AAV serotypes (Fig. 4). In the cross-sections, EGFP expression was limited mainly to the photoreceptor outer segment (POS) with AAV-2 injections. In contrast, the expression spread into the entire photoreceptor layer, and a few signals in the inner retinal layers also were seen with AAV-DJ injections (Figs. 4D–4G).

Functional changes after AAV injection

To examine changes in retinal function associated with AAV infection, electroretinography (ERG) was performed. The scotopic ERG showed a slight but non-significant reduction in the b-wave amplitude with injection of vehicle (n = 6 eyes), AAV-2 (n = 6 eyes), or AAV-DJ (n = 6 eyes) compared to age-matched controls without injections (n = 6 eyes). However, no significant difference was observed between the injection groups (Fig. 5). These data indicated that the reduction in the ERG amplitude was not due to infectious toxicity of AAV but invasion of the intravitreal injection itself.