The focus of this study was to determine whether a suppression and replacement gene therapy involving cosubretinal administration of two AAV vectors, one encoding a RHO suppressor (AAV-S) and the other a RHO replacement gene (AAV-R), could provide significant benefit in a mouse model of RHO-adRP, the P347S mouse.
The working hypothesis was that coadministration of two AAVs (AAV-S and AAV-R) would result in coinfection of significant proportions of photoreceptors. In principle, these photoreceptors should receive both suppression and replacement components and therefore should function similarly to wild-type photoreceptors. To test the feasibility of this approach, coadministration of two AAV vectors each encoding a marker gene was undertaken. Both reporter genes were driven from a CMV promoter. A mixture of 1.5 × 109 vector particles (vp) AAV-EGFP and 1.5 × 109 vp AAV-DsRed was subretinally injected into adult wild-type mice and retinas evaluated by histology 2 weeks later (n = 5). Areas of the retinas transduced by the two viruses completely overlapped as determined by microscopic analysis of whole mount retinas (). At the cellular level the majority of the transduced cells, coexpressed both markers (yellow) and only a few cells expressed just one marker (red or green; ). These experiments provide evidence of significant coexpression following coadministration of two AAVs. The results suggest that a similar strategy using two AAV constructs for delivering the suppression and replacement components should also result in significant cotransduction of photoreceptors.
Figure 1 Cotransduction of adeno-associated viruses (AAVs) in wild-type retinas. Eyes of wild-type mice were subretinally injected with a mixture of 1.5 × 109 vector particles (vp) AAV-EGFP and 1.5 × 109 vp AAV-DsRed. Two weeks postinjection eyes (more ...)
Initially, 6.0 × 108
vp AAV-S or an AAV expressing a nontargeting control RNAi, AAV-C, were subretinally injected into contralateral eyes of P347S
mouse pups, which express a human mutant RHO
transgene. AAV-S has previously been shown to suppress rhodopsin mRNA by >90% in vivo
(also referred to as siBB,13
). Two weeks postinjections retinas were harvested, transduced (green) cells collected by fluorescence-activated cell sorting and RNA extracted. Levels of mutant RHO
, determined by quantitative real-time reverse transcription PCR (qPCR), using human RHO
-specific primers, were suppressed by 68 ± 2.4% (n
= 6, P
= 0.0187) in cells transduced with AAV-S versus AAV-C, indicating that efficient RHO
suppression has occurred in this disease model of RHO-adRP (
). Subsequently the resistance to suppression of transcripts expressed from AAV-R was determined in adult wild-type mice. Mixtures of either 6.0 × 108
vp AAV-S and 1.8 × 1010
vp AAV-R or 6.0 × 108
vp AAV-C and 1.8 × 1010
vp AAV-R were subretinally injected into fellow eyes, total RNA extracted 2 weeks postinjection and RNA analyzed. Levels of RHO
replacement expressed from AAV-R were determined by qPCR and did not differ significantly between the AAV-S and AAV-R or the AAV-C and AAV-R injected eyes (P
= 0.814, n
= 7), suggesting that the RHO
replacement gene delivered in AAV-R is resistant to AAV-S suppression (
). Additionally, subretinal administration of 1.0 × 1010
vp of AAV-R was undertaken in adult wild-type mice (n
= 8) and 2 weeks postinjection levels of RHO
expression from AAV-R were compared to levels of expression of RHO
in normal human rhodopsin (NHR
) transgenic mice. The NHR
mouse expresses a wild-type human RHO
transgene at levels comparable with endogenous levels of mouse Rho
expression and displays a wild-type phenotype.13,17
Comparison of RNA from whole retinas of AAV-R administered to wild-type mice and retinas from NHR
mice demonstrated that RHO
expression from AAV-R was ~31 ± 5% of levels observed in NHR
mouse retinas. Notably, since only ~40% of the retina is thought to be transduced by AAV (
), this suggests that overall expression levels of RHO
from AAV-R may be similar to endogenous murine Rho
levels and RHO
levels in the NHR
transgenic mouse (
). Quantitative protein analysis was not undertaken as the human RHO-specific antibody used for immunocytochemistry (see below) is not suitable for western blotting or enzyme-linked immunosorbent assay.
Figure 2 Rhodopsin mRNA expression levels. (a) Contralateral eyes of adult P347S mice (n = 6) expressing a mutant human RHO transgene were subretinally injected with 6.0 × 108 vp AAV-S or AAV-R. Two weeks postinjection retinas were harvested, transduced (more ...)
In all subsequent experiments, 5-day-old P347S mice were subretinally injected. In one set of experiments, right eyes were injected with a mixture of 6.0 × 108 vp AAV-S and 1.8 × 1010 vp AAV-R. Fellow left eyes received 6.0 × 108 vp of control AAV-C. As evaluated by 6-week postinjection ERGs, rod-isolated amplitudes in eyes treated with the mixture of AAV-S and AAV-R were found to be 184.5 ± 65.4 µV compared to fellow eyes treated with control AAV-C, whereas the amplitudes were 34.9 ± 16.8 µV (P <0.0001, n = 17; ). In 20-week postinjection ERGs, rod-isolated responses were 58.1 ± 19.8 µV in treated eyes compared to 16.9 ± 12.6 µV (P < 0.0001, n = 12) in control eyes (), the latter being similar to that of uninjected eyes (data not shown).
Figure 3 Rod-derived electroretinography (ERG) following combined suppression and replacement therapy. The right eyes of P5 P347S mice were subretinally injected with a mixture of 6.0 × 108 vp AAV-S and 1.8 × 1010 vp AAV-R whereas the left eyes (more ...)
In order to determine whether the improved retinal responses resulted from the AAV-S and AAV-R suppression and replacement combination therapy or either component singly, effects of AAV-R and AAV-S were assessed separately. To test the suppression component, 6.0 × 108 vp of AAV-S (right eyes) or control AAV-C (left eyes) were subretinally injected into contralateral eyes. Similarly, to test the replacement component, 1.8 × 1010 vp of AAV-R (right eyes) were subretinally injected whereas the fellow left eyes remained uninjected. Rod-isolated ERGs, performed 6 weeks postinjection were not significantly different; 60.5 ± 32.6 µV in AAV-S alone treated eyes compared to 68.1 ± 20.1 µV (n = 12) in control eyes (Supplementary Figure S1a,c) and 63.7 ± 38.6 µV in AAV-R alone treated eyes compared to 46.4 ± 17.9 µV (n = 10) in uninjected eyes (Supplementary Figure S1b,c) indicating that AAV-S alone or AAV-R alone did not provide benefit in P347S mice.
Notably, the benefit observed subsequent to subretinal delivery of the combined suppression and replacement therapy was also observed at the histological level. Six weeks postinjection, outer nuclear layer (ONL) thickness of sections from eyes treated with the mixture of AAV-R and AAV-S was 17.9 ± 3.4 µm compared to 13.3 ± 2.0 µm in sections from control eyes treated with AAV-C (P <0.0001, n = 5). Preservation of retinal structure was also apparent in semithin sections (Supplementary Figure S2a,b). Transmission electron microscopy (TEM) of these retinas (n = 3) demonstrated that only photoreceptor inner segments were present in control retinas whereas photoreceptor inner segments and OS were abundant in AAV-S- and AAV-R-treated retinas (Supplementary Figure S2c,d). Due to the presence of OS, the distance between the ONL and the retinal pigment epithelium (RPE) was greater in the treated eyes (Supplementary Figure S2).
Histological analysis at 20 weeks postinjection demonstrated a striking difference between combined suppression and replacement-treated and control eyes. Whereas vestigial ERG responses were still recorded in some control eyes, the ONL had almost completely disappeared (and therefore ONL thickness was not measurable) and rhodopsin protein was not detectable in the control eyes (). In contrast, the ONL in eyes treated with suppression and replacement therapy contained 3–4 layers of photoreceptor nuclei (8.9 ± 1.2 µm thickness; n = 4, ). Treated retinas were characterized by rhodopsin expression in both the ONL and the photoreceptor segment layer (). Note that enhanced green fluorescent protein (EGFP) tracer was only present in the RPE of the control eyes (as no photoreceptor layer remained; ) whereas it was present in both RPE and ONL of the suppression- and replacement-treated retinas (). Differences in EGFP intensities between treated and control eyes were apparent in the whole mount retinas () and were independent of the transduction coverage (~40%). Semithin sections demonstrated significant photoreceptor rescue in the treated retinas (). At the ultrastructural level, well-preserved OS characterized the treated retina () whereas only membranous debris was present between the inner nuclear layer and the RPE () in the control retina. High magnification TEM revealed individual photoreceptor segments, depicted in . Short, degenerating OS with inflated and disorganized membrane disks were present in the control retina at 6 weeks postinjection (), whereas no photoreceptors were found at 20 weeks postinjection. In photoreceptor cells treated with AAV-S and AAV-R, the inner segments were attached to well-preserved OS with parallel layers of tightly stacked membrane disks at both 4 and 20 weeks postinjection indicating substantial rescue of the photosensitive OS ().
Figure 4 Immunohistochemical analysis of rhodopsin expression following combined suppression and replacement therapy 20 weeks postinjection. The right eyes of P5 P347S mice were subretinally injected with a mixture of (b,d, and f) 6.0 × 108 vp AAV-S and (more ...)
Figure 5 Ultrastructural analysis of combined suppression and replacement-treated retinas 20 weeks postinjection. The right eyes of P5 P347S mice were subretinally injected with a mixture of (b,d) 6.0 × 108 vp AAV-S and 1.8 × 1010 vp AAV-R whereas (more ...)
Figure 6 Photoreceptor morphology rescue following combined suppression and replacement therapy. The right eyes of P5 P347S mice were subretinally injected with a mixture of 6.0 × 108 vp AAV-S and 1.8 × 1010 vp AAV-R (b and c; n = 3 and n = 1, (more ...)