Lentiviral Expression of Retinal Guanylate Cyclase-1 (RetGC1) Restores Vision in an Avian Model of Childhood Blindness

BACKGROUND: Leber congenital amaurosis (LCA) is a genetically heterogeneous group of retinal diseases that cause congenital blindness in infants and children. Mutations in the GUCY2D gene that encodes retinal guanylate cyclase–1 (retGC1) were the first to be linked to this disease group (LCA type 1 [LCA1]) and account for 10%–20% of LCA cases. These mutations disrupt synthesis of cGMP in photoreceptor cells, a key second messenger required for function of these cells. The GUCY1*B chicken, which carries a null mutation in the retGC1 gene, is blind at hatching and serves as an animal model for the study of LCA1 pathology and potential treatments in humans. METHODS AND FINDINGS: A lentivirus-based gene transfer vector carrying the GUCY2D gene was developed and injected into early-stage GUCY1*B embryos to determine if photoreceptor function and sight could be restored to these animals. Like human LCA1, the avian disease shows early-onset blindness, but there is a window of opportunity for intervention. In both diseases there is a period of photoreceptor cell dysfunction that precedes retinal degeneration. Of seven treated animals, six exhibited sight as evidenced by robust optokinetic and volitional visual behaviors. Electroretinographic responses, absent in untreated animals, were partially restored in treated animals. Morphological analyses indicated there was slowing of the retinal degeneration. CONCLUSIONS: Blindness associated with loss of function of retGC1 in the GUCY1*B avian model of LCA1 can be reversed using viral vector-mediated gene transfer. Furthermore, this reversal can be achieved by restoring function to a relatively low percentage of retinal photoreceptors. These results represent a first step toward development of gene therapies for one of the more common forms of childhood blindness

Comparison of Retinal Morphology of Wild-Type RIR, Untreated GUCY1*B, and Treated GUCY1*B Chickens

<div><p>(A) The morphology of the retinas of treated animals was examined to determine if the viral treatment had affected the course of retinal degeneration. Regions were analyzed along the vertical meridian of the right eyes of experimental and control animals; representative micrographs from the locus superior to the optic nerve (SUP ON) are shown from one wild-type, one untreated GUCY1*B, and three treated retinas (animals 1, 5, and 6). GCL, ganglion cell layer; INL, inner nuclear layer; IPL, inner plexiform layer; IS, inner segments; ONL, outer nuclear layer; OS, outer segments; RPE, retinal pigment epithelium.</p> <p>(B) Relative percent change in retinal thickness of treated animals. Retinal width (in micrometers) from the outer limiting membrane to the ganglion cell layer was measured at six loci (100 μm apart) within the regions examined. These widths were expressed as percent change relative to average widths of the untreated GUCY1*B retinas in these regions, which were set to zero for graphing purposes. A simple one-sample <i>t</i>-test (null hypothesis that treatment groups are not different from untreated GUCY1*B) was used to analyze the results. The results of this test showed that retinal thicknesses of the treated animals in the SUP ON (SON) and INF ON (ION) regions were greater than those of untreated animals ( <i>p</i> < 0.05). Four of six treated animals also showed evidence of slowing of degeneration in the SUP region, but this was not statistically significant. The key for animal number is shown in the upper left of the graph. </p></div

Immunohistochemical Analyses of GFP Expression in Treated Retinas

<div><p>(A) Schematic of the right eye cup that shows how the retina was apportioned for analyses. The right eye of each animal was bisected along the vertical meridian. One half was processed for immunohistochemical (IHC) analyses and the other for detailed histological analyses. The retinal sections were divided into four regions (FAR SUP, SUP, SUP ON, and INF ON) to simplify analyses.</p> <p>(B) Flat-mount retinas stained to reveal pattern of transduction obtained following neural tube delivery of 0.5 μl of EF1α-PLAP virus (10⁹ TUs/ml). The percent transduction for the left (left panel) and right (right panel) eyes of two of the three animals analyzed is shown. </p> <p>(C) FAR SUP region of a treated retina immunostained for GFP. Arrows indicate staining in photoreceptor cell bodies. GFP staining is also visible in the inner segments and outer segments of these cells. GCL, ganglion cell layer; INL, inner nuclear layer; IPL, inner plexiform layer; IS, inner segments; ONL, outer nuclear layer; OS, outer segments.</p> <p>(D) Topographical distribution of GFP-expressing cells in treated right eyes from treated animals 2 (left) and 1 (right). The percent transduction of each of the four retinal regions was plotted on the <i>z</i>-axis as a function of location along the superior–inferior axis of the eye ( <i>x-</i>axis) and distance from the midline ( <i>y</i>-axis). These analyses represent the results obtained from serial sections over 500 μm beginning at the midline axis and moving laterally. </p></div

In Vitro Analyses of the Function of the pTYF-EF1α-GC1-IRES-eGFP Vector and Virus

<div><p>(A) Diagram of the bicistronic vector indicating production of bovine GC1 and GFP proteins from a single transcript.</p> <p>(B) DF1 cells transiently transfected with the vector and subsequently analyzed for expression of bovine GC1 and GFP. Cells immunostained with antibody recognizing bovine GC1 also expressed GFP.</p> <p>(C) Comparison of GC1 activity measured in bovine rod outer segments (ROS), TE671 cells transduced with EF1α-GC1-IRES-eGFP virus, and TE671 cells transduced with the control virus, EF1α-eGFP. Activity was assayed in the presence or absence of bovine GCAP1 under both high- and low-calcium conditions and is expressed as nanomoles of cGMP produced per minute.</p> <p>(D) Examination of ability of chicken GCAP1 to activate bovine GC1 under physiological conditions. GC1 activity was measured in preparations of bovine rod outer segments in the presence and absence of bovine GCAP1 and chicken GCAP1 under high- and low-calcium conditions. All assays were conducted in triplicate. Bars represent mean ± SEM.</p></div

Optokinetic and Volitional Behavior Tests Indicate Treatment Efficacy

<div><p>(A) Optokinetic reflex (OKN) exhibited by 21-d-old, wild-type RIR chicken. Two frames of <a href="http://www.plosmedicine.org/article/info:doi/10.1371/journal.pmed.0030201#sv001" target="_blank">Video S1</a> (4.3 s between frames) are shown illustrating the head movement observed in birds in response to counterclockwise rotation of the 0.26-cycles·degree⁻¹ (bar width = 1.25 cm) stimulus. </p> <p>(B) Volitional visual behavior exhibited by treated GUCY1*B animal 2 on day 7. Two frames of <a href="http://www.plosmedicine.org/article/info:doi/10.1371/journal.pmed.0030201#sv004" target="_blank">Video S4</a> are shown illustrating the animal's abilities to perceive and peck at objects within its visual field. </p> <p>(C and D) Graphic summaries of the behavioral test results for the six treated animals that exhibited sighted behavior. (C) Graphs showing the optokinetic scores (0–3) for the six treated animals as a function of age. (D) Graphs showing the volitional behavior scores (0–3) for the six treated animals as a function of age. The graphs are color coded by animal.</p></div

Quantitative Genomic PCR Estimate of Integrated Viral Trangenes per Genome

<div><p>(A) Diagram of integrated viral transgene and location of PCR primers spanning the IRES-eGFP junction that were used to amplify the transgene.</p> <p>(B) Ethidium-bromide-stained gel showing 638-bp product (indicated by arrow) amplified from the experimental and standard reactions. Lanes 1–6 contain samples amplified from the six treated animals exhibiting restored sight. No product was observed in untreated GUCY1*B (lanes 7 and 8) or wild-type (lane 9) chickens. A blank water control is shown in lane 10. Lanes 11–14 contain product amplified from 300,000, 150,000, 30,000, and 15,000 copies of the transducing vector. DNA size ladders, 1 kb and 123 bp, were run to the left of the samples and standards.</p> <p>(C) Plot of amount of product obtained from the standard PCR reactions (black) best fit with a sigmoid curve ( <i>f</i> = <i>y</i>₀ + <i>a</i>/(1 + exp(−( <i>x −x</i>₀)/ <i>b</i>)) <i>^c</i> with <i>r</i>² = 0.999 . The products obtained from each experimental sample (red) fell within the linear portion of the amplification curve, and the number of transgenes present in each sample was calculated using the equation for the best-fit curve. Individual values plotted are mean ± SEM ( <i>n</i> = 3). </p> <p>(D) Comparisons of integrated viral transgenes per genome, dark-adapted ERG b-wave amplitudes (1 Hz), volitional behavior scores, and optokinetic reflex scores. The data for the ERG and behavioral tests were collected at the end of the 6-wk study period. The results for the six treated animals exhibiting sight and for age-matched untreated GUCY1*B and wild-type RIR animals are shown. NA, not assayed; ND, not detected.</p></div

Retinal Electrophysiology Rescued in Treated Eyes

<div><p>(A) Comparison of dark-adapted ERGs in response to increasing intensities of light in a GUCY1*B/GUCY1*B animal injected with EF1α-bGC1-IRES-eGFP (“treated”) compared to a control animal (“untreated”). ERGs in the untreated animal are nondetectable, in contrast to the sizeable ERGs evoked in the treated animal. Results from a wild-type animal are shown in the left column for comparison.</p> <p>(B) Light-adapted 29-Hz flicker ERGs in the same animals as shown in (A) demonstrate restoration of responses after treatment.</p> <p>(C) Overlapping waveforms are ERGs elicited by 0.8 log cd·s·m⁻² white flashes presented in dark-adapted (“DA 1 Hz”) and light-adapted (“LA Flicker”) states in all treated animals compared to wild-type controls. Functional rescue was observed in five (top waveforms) of the seven treated animals, whereas two (bottom waveforms) showed responses indistinguishable from noise. </p> <p>(D) Summary parameters of dark-adapted photoreceptor (a-wave) and post-photoreceptor (b-wave) function, as well as light-adapted flicker amplitude in treated animals as compared to untreated and wild-type animals. Five of seven treated animals showed amplitudes substantially larger than untreated animals but smaller than wild-type controls (gray symbols; mean ± standard deviation).</p></div

Long-term Retinal Function and Structure Rescue Using Capsid Mutant AAV8 Vector in the rd10 Mouse, a Model of Recessive Retinitis Pigmentosa

The retinal degeneration 10 (rd10) mouse is a well-characterized model of autosomal recessive retinitis pigmentosa (RP), which carries a spontaneous mutation in the β subunit of rod cGMP-phosphodiesterase (PDEβ). Rd10 mouse exhibits photoreceptor dysfunction and rapid rod photoreceptor degeneration followed by cone degeneration and remodeling of the inner retina. Here, we evaluate whether gene replacement using the fast-acting tyrosine-capsid mutant AAV8 (Y733F) can provide long-term therapy in this model. AAV8 (Y733F)-smCBA-PDEβ was subretinally delivered to postnatal day 14 (P14) rd10 mice in one eye only. Six months after injection, spectral domain optical coherence tomography (SD-OCT), electroretinogram (ERG), optomotor behavior tests, and immunohistochemistry showed that AAV8 (Y733F)-mediated PDEβ expression restored retinal function and visual behavior and preserved retinal structure in treated rd10 eyes for at least 6 months. This is the first demonstration of long-term phenotypic rescue by gene therapy in an animal model of PDEβ-RP. It is also the first example of tyrosine-capsid mutant AAV8 (Y733F)-mediated correction of a retinal phenotype. These results lay the groundwork for the development of PDEβ-RP gene therapy trial and suggest that tyrosine-capsid mutant AAV vectors may be effective for treating other rapidly degenerating models of retinal degeneration