Leber congenital amaurosis type 2 (LCA2) is an inherited disease that affects the
integrity of the retina and results in severe visual impairment early in life [1].
This disease is caused due to mutations in the retinal pigmental epithelium (RPE)
65 kDa protein encoding gene, that generates an isomerohydrolase enzyme in the visual
cycle [2]. Gene therapy is an attractive option for this condition, due to the relatively
immune privileged nature of the eye. Adeno-associated virus serotype 2 (AAV2) vectors
have been utilized to deliver RPE65 gene into LCA2 patients [3]. The long-term follow-up
data [4] demonstrated a peak functional rescue, ~1 year after gene therapy, but subsequently
a decline in RPE65 expression and immune response was noted [5]. Thus, AAV2 vectors
that can augment visual function at significantly lower doses are needed. In our recent
study [6], we have identified and demonstrated the role of ubiquitin-like modifiers
such as Neddylation in AAV2 vectors and abolition of these sites, augmented coagulation
factor IX expression in hemophilia B mice.
The present study was designed to evaluate if these Neddylation-site modified AAV2
vectors are effective, during ocular gene therapy. To assess this, we packaged wild-type
(WT) AAV2 vector (ssAAV2-RPE65; scAAV2-CB-EGFP) and a Neddylation-mutant vector containing
a human RPE65 gene or the enhanced green fluorescent protein (EGFP) gene (ssAAV2-K665Q-RPE65;
scAAV2-K665Q-CB-EGFP), as described earlier [6]. Vector titers were measured by a
quantitative PCR and are expressed as vector genomes (vgs)/ml. All the animal experiments
were approved by the IIT-Kanpur animal ethics committee. The AAV vectors thus generated
were assessed by in vivo ocular gene transfer by different routes of delivery (intravitreal
and subretinal) and strains of mice [C57BL6/J and rd12, Jackson Laboratory (Bar Harbor,
USA)]. In the first set of investigations, eyes (n = 8 per group) of C57BL6/J mice
were either mock injected or injected with AAV2-WT-EGFP and AAV2-K665Q-EGFP vectors
by the intravitreal or subretinal route at a dose of 3 × 108 vgs/eye. Fluorescence
imaging of the eyes was performed, 2 and 8 weeks after ocular gene transfer in a Micron
IV imaging system (Phoenix Research Lab, Pleasanton, USA). Our data shown in Fig. 1a,
demonstrate that the K665Q mutant had a significantly higher EGFP expression (7.87–9.72-fold,
p < 0.05, Fig. 1b) when compared with eyes that were administered with AAV2-WT vectors,
intravitreally.
Fig. 1
Ocular gene transfer in C57BL6/J and rd12 mice with a Neddylation-site modified K665Q
vector.
Eyes of C57BL6/J mice (n = 8) were mock injected or injected with scAAV2-EGFP and
scAAV2-K665Q-EGFP vectors at a dose of 3 × 108 vgs by either intravitreal or subretinal
route. a Fundus imaging of the murine eyes was performed 2 and 8 weeks after intravitreal
administration in a Micron IV imaging system (Phoenix research labs, CA, USA). Representative
images from 8-week imaging data are shown. Intensity was set at maximum and gain was
set at 15 db, frame rate was set at 6 fps, for imaging of all the groups. b A quantification
of the data obtained was performed by using Concentric Circle Plugin in the ImageJ
software. c Murine eyes administered via subretinal administration with AAV vectors
were enucleated after 4 weeks and 8 µM cryosections were obtained. Sections of mock
injected, scAAV2-EGFP, and scAAV2-K665Q-EGFP groups were counterstained for nuclei,
with 4′,6-diamidino-2-phenylindole (DAPI). Fluorescence images were acquired by A1R
HD25 Nikon Confocal (Ti-2 eclipse body) microscope (Tokyo, Japan) with a 405 nm and
488 nm laser equipped with Galvano scanners. Representative images are shown. d Eyes
of rd12 mice (n = 6) were mock- injected or injected with ssAAV2-RPE65 and ssAAV2-K665Q-RPE65
vectors at a dose of 7 × 108 vgs, via subretinal route. Scotopic electroretinography
was performed after 16 weeks by a Ganzfeld ERG system (Phoenix research labs, CA,
USA). Representative ‘A-wave’ and ‘B-wave’ forms are shown (d) along with the quantitative
data (e). Data shown are mean ± SD. ANOVA based Dunett’s test was used for statistical
comparison between the groups. **p < 0.01, ***p < 0.001. f, g For immunostaining,
8 µM cryosections of mock injected, ssAAV2-RPE65 and ssAAV2-K665Q-RPE65 groups were
stained with anti-RPE65 (1:100, Abcam, Cambridge, UK) and counterstained with goat
anti-mouse Alexa Fluor™ 555 (1:300, Abcam) antibody or with anti-GFAP (1:100, Cell
signaling technologies, Danvers, USA) and counterstained with goat anti-rabbit cy3
antibody (1:300, Jackson ImmunoResearch, West Grove, USA). Images were acquired in
Leica DMi8 confocal microscope (Wetzlar, Germany) with a 405 nm and 532 nm laser equipped
with Galvano scanners. A representative set of DAPI overlay images with RPE65 (f)/GFAP
(g) staining are shown. GCL ganglion cell layer, INL inner nuclear layer, ONL outer
nuclear layer; OS outer segment; RPE retinal pigment epithelium (marked with dotted
line in (f)).
We then examined the transduction potential of the AAV2-K665Q-EGFP vector by subretinal
administration. Four weeks later, retinal sections (8 µm) of the murine eyes was prepared,
stained with DAPI (Sigma-Aldrich, St. Louis, USA) and mounted with FluorSave™ (Sigma-Aldrich).
Images were acquired in a confocal microscope (A1R HD25 Nikon, Tokyo, Japan). Our
analysis of K665Q-EGFP vector administered eyes, revealed a markedly enhanced GFP
expression within the RPE layer and the outer segment of the retina in comparison
to AAV2-WT vector (Fig. 1c).
To further evaluate the therapeutic efficiency of the mutant AAV2 vectors in a murine
model of retinal degeneration, we administered either PBS (mock group), AAV2-WT, and
AAV2-K665Q vectors expressing human RPE65, in rd12 mice. Approximately, 1–2 µl of
AAV vectors at dose of 7 × 108 vgs was administered via subretinal route into the
murine eyes (n = 6 eyes per group). The phenotypic response was measured by scotopic
electroretinography (ERG), 16 weeks after vector administration (Ganzfeld ERG, Phoenix
Research lab). The representative ERG waveforms from the treated mice are shown in
Fig. 1d. We noted a significant visual correction in eyes that received AAV2-K665Q-RPE65
vectors, with a 2.43-fold (p < 0.001) increase in ‘A-wave’ amplitude and a 1.25-fold
(p < 0.01) increase in ‘B-wave’ amplitude in comparison to the AAV2-WT injected animals
(Fig. 1d, e), at the very low doses of AAV vectors administered in this study (7 × 108
vgs/eye). Also, immunostaining of vector injected eye sections with an anti-RPE65
antibody (Abcam, Cambridge, UK) revealed enhanced RPE65 expression in AAV2-K665Q administered
animals (Fig. 1f). Further, the expression of glial fibrillary acidic protein (GFAP)
was similar between the treatment groups suggesting the lack of inflammation in treated
eyes (Fig. 1g).
Our preclinical data presented here highlights the translational potential of Neddylation-site
modified vectors for retinal gene therapy. However, further long-term follow-up data
and a comprehensive evaluation of ocular immune response in vivo are required prior
to its clincial application.