Rescue of inherited retinal degeneration associated with Aipl1 defects

Since groundbreaking clinical trials treating Leber congenital amaurosis (LCA) patients with retinal pigmented epithelium-specific 65 kDa protein (Rpe65) defects, which were the most successful gene therapy studies to date, much effort has been put into finding other candidate genes associated with inherited retinal degeneration to treat. Animal models with Rpe65 defects demonstrate an unusual circumstance that greatly favors treatment, where dramatic vision loss is disproportionately greater than the ensuing slow photoreceptor degeneration. This pattern is also believed to occur in Rpe65-LCA patients, and stems from the primary defect in retinal pigmented epithelial (RPE) cells that subsequently perturbs photoreceptors. However, a majority of inherited retinal degenerations are associated with photoreceptor gene defects, and do not show this advantageous disparity between visual dysfunction and photoreceptor loss. To combat this, efforts have concentrated on improving photoreceptor transduction efficiency with viral vectors, and delivering gene replacement to slower progressing retinal dystrophies.;Aryl hydrocarbon receptor interacting protein-like1 (Aipl1) is mainly associated with the severe retinal degeneration, LCA. However, reports of Aipl1 defects in patients showing slower progressing retinal disease, retinitis pigmentosa (RP) and cone-rod dystrophy (CORD), points to a subset of Aipl1 patients as potential candidates for future AAV-mediated gene therapy trials. Multiple mouse models with Aipl1 defects have been developed and contribute to the ease of conducting pre-clinical rescue studies to examine the rescue potential of Aipl1..;To investigate the rescue potential of Aipl1 defects, we conducted AAV-mediated gene replacement studies in Aipl1-null mice, a model for Aipl1-LCA, the most rapidly progressing of any retinal degeneration mouse model. Our study examines the recent advancements in AAV viral vectors, using a combined self-complementary Y733F capsid mutant AAV8 (sc-Y733F-AAV8), to evaluate whether these advancements would provide functional benefits to a model of retinal degeneration. We found that the sc-Y733F-AAV8 viral vector significantly improved vision rescue in Aipl1-null mice as compared to conventional single-stranded AAV8, even when treatment was administered at postnatal day 10, after initiation of photoreceptor degeneration. From these studies, we also conclude that the Aipl1-null mouse provides a good model to test the functional benefits of ongoing and future advancements in AAV viral vectors.;In addition to LCA, Aipl1 has been reported in patients with CORD and RP. We were particularly interested in a mutation, P351Delta12 hAipl1 because of its association with CORD and our ongoing interest in the role of Aipl1 in cone photoreceptors. We generated a transgenic mouse model expressing P351Delta12 hAIPL1, which showed a cone-rod dystrophy phenotype similar to a case of Aipl1-CORD in a patient with this mutation. The mice demonstrated early and rapidly declining cone-mediated vision with slow progressing rod defects. Additionally, in our studies of double transgenic mice expressing both mutant P351Delta12 hAIPL1 and WT hAIPL1, we conclude that the P351Delta12 hAipl1 mutation does exert dominant effects that explain the autosomal dominant inheritance pattern observed in the Aipl1 patients with this mutation.;Interestingly, despite the dominant phenotype, we were able to rescue cone dysfunction and degeneration following AAV-mediated gene delivery of WT hAIPL1. We believe this is due to the drastic overexpression of WT hAIPL1 that is achieved with AAV gene delivery. Further work with this novel model of Aipl1-CORD is needed to elucidate the dominant mechanism of the P351Delta12 hAIPL1 protein, which appear to behave differently in rods and cones, as rod dysfunction was not rescued with AAV-mediated overexpression of WT hAIPL1. With these successes in pre-clinical Aipl1 rescue studies, which have also been independently demonstrated by two other groups, we conclude that Aipl1 defects show great potential for movement into AAV-mediated gene therapy clinical trials

Granulicatella adiacens, an unusual causative agent in chronic dacryocystitis

West Virginia Universit

Perimacular Atrophy Following Voretigene Neparvovec-Rzyl Treatment in the Setting of Previous Contralateral Eye Treatment With a Different Viral Vector

PurposeTo report on cases of unilateral perimacular atrophy after treatment with voretigene neparvovec-rzyl, in the setting of previous contralateral eye treatment with a different viral vector.DesignSingle-center, retrospective chart review.MethodsIn this case series, four patients between the ages of six and 11 years old with RPE65-related retinopathy were treated unilaterally with rAAV2-CB-hRPE65 as part of a gene augmentation clinical trial (NCT00749957). Six to 10 years later the contralateral eyes were treated with the Food and Drug Administration-approved drug, voretigene neparvovec-rzyl. Best-corrected visual acuity (BCVA), fundus photos, ocular coherence tomography, two-color dark-adapted perimetry, full field stimulus threshold testing (FST), and location of subretinal bleb and chorioretinal atrophy were evaluated.ResultsThree out of four patients showed unilateral perimacular atrophy after treatment with voretigene, ranging from five to 22 months after treatment. Areas of robust visual field improvement were followed by areas of chorioretinal atrophy. Despite perimacular changes, BCVA, FST, and subjective improvements in vision and nyctalopia were maintained. Perimacular atrophy was not observed in the first eye treated with the previous viral vector.ConclusionsWe observed areas of robust visual field improvement followed by perimacular atrophy in voretigene treated eyes, as compared to the initially treated contralateral eyes.Translational relevanceCaution is advised when using two different viral vectors between eyes in gene therapy. This may become an important issue in the future with increasing gene therapy clinical trials for inherited retinal dystrophies

Retinal Neuroprotective Effects of Flibanserin, an FDA-Approved Dual Serotonin Receptor Agonist-Antagonist

Purpose: To assess the neuroprotective effects of flibanserin (formerly BIMT-17), a dual 5-HT1A agonist and 5-HT2A antagonist, in a light-induced retinopathy model. Methods: Albino BALB/c mice were injected intraperitoneally with either vehicle or increasing doses of flibanserin ranging from 0.75 to 15 mg/kg flibanserin. To assess 5-HT1A-mediated effects, BALB/c mice were injected with 10 mg/kg WAY 100635, a 5-HT1A antagonist, prior to 6 mg/kg flibanserin and 5-HT1A knockout mice were injected with 6 mg/kg flibanserin. Injections were administered once immediately prior to light exposure or over the course of five days. Light exposure lasted for one hour at an intensity of 10,000 lux. Retinal structure was assessed using spectral domain optical coherence tomography and retinal function was assessed using electroretinography. To investigate the mechanisms of flibanserin-mediated neuroprotection, gene expression, measured by RT-qPCR, was assessed following five days of daily 15 mg/kg flibanserin injections. Results: A five-day treatment regimen of 3 to 15 mg/kg of flibanserin significantly preserved outer retinal structure and function in a dose-dependent manner. Additionally, a single-day treatment regimen of 6 to 15 mg/kg of flibanserin still provided significant protection. The action of flibanserin was hindered by the 5-HT1A antagonist, WAY 100635, and was not effective in 5-HT1A knockout mice. Creb, c-Jun, c-Fos, Bcl-2, Cast1, Nqo1, Sod1, and Cat were significantly increased in flibanserin-injected mice versus vehicle-injected mice. Conclusions: Intraperitoneal delivery of flibanserin in a light-induced retinopathy mouse model provides retinal neuroprotection. Mechanistic data suggests that this effect is mediated through 5-HT1A receptors and that flibanserin augments the expression of genes capable of reducing mitochondrial dysfunction and oxidative stress. Since flibanserin is already FDA-approved for other indications, the potential to repurpose this drug for treating retinal degenerations merits further investigation

Flibanserin’s neuroprotective effects are 5-HT_1A receptor-mediated.

<p>(<b>A i</b>) A spider graph representing average right-eye receptor plus values demonstrates that pre-treatment with WAY 100635 prior to 6 mg/kg flibanserin (10 mg/kg WAY + 6 mg/kg Flibanserin, <i>green</i>) decreased average receptor plus thickness as compared to flibanserin-treatment without WAY 100635 (6 mg/kg Flibanserin, <i>blue</i>). A 6 mg/kg dose of flibanserin in 5-HT_1A knockout mice (5-HT1A KO + 6 mg/kg Flibanserin, <i>red</i>) resulted in average receptor plus thickness that was not significantly different from vehicle-injected and naïve mice. The gray area indicates ± 2 SD of the naïve averaged data. (<b>A ii, A iii</b>) Receptor plus thicknesses, with each dot representing average right and left eye thickness from one mouse, demonstrate that both pre-treatment of BALB/c mice with WAY 100635 prior to a flibanserin injection (10 mg/kg WAY + 6 mg/kg Flibanserin, <i>green</i>) and a flibanserin injection in 5-HT_1A knockout mice (5-HT1A KO + 6 mg/kg Flibanserin, <i>red</i>) significantly reduces the observed neuroprotective effects provided by a single 6 mg/kg dose of flibanserin (6 mg/kg Flibanserin, <i>blue</i>) in the <b>(A ii)</b> temporal quadrant and the <b>(A iii)</b> nasal quadrant. (<b>B</b>) Pre-treatment with WAY 100635 (10 mg/kg WAY + 6 mg/kg Flibanserin, <i>green</i>) mitigated the improvements to ERG a- and b-wave amplitudes provided by a single 6 mg/kg dose of flibanserin (<i>blue</i>). ERG a- and b-wave amplitudes were not improved by a single dose of 6 mg/kg flibanserin when administered to 5-HT_1A knockout mice (5-HT1A KO + 6 mg/kg Flibanserin, <i>red</i>). “†” indicates the flash intensity providing the b_(max,rod) value at which statistical analyses was also performed and presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0159776#pone.0159776.s001" target="_blank">S1C Fig</a>. * indicates a significant difference from both the vehicle-treated group and the naïve group, <i>P</i> < 0.05. n.s. indicates non-significance with <i>P</i> > 0.05.</p

A 5-day time course of flibanserin preserves retinal function in a dose-dependent manner, as measured by ERG.

<p>(<b>A</b>) Representative ERG traces at the 3.55 log cd•s/m² light intensity demonstrating dose-dependent improvements in ERG responses as compared to vehicle-injected mice. (<b>B</b>) Mice treated with doses of 3 mg/kg or greater of flibanserin showed significantly higher ERG b-wave and a-wave responses at the highest ERG flash intensity (3.55 log cds/m²) as compared to vehicle-treated mice. Individual ERGs were averaged with ERGs for mice within its respective group and are represented as mean ± standard error. * indicates a significant difference from both the vehicle-treated group and the naïve group, <i>P</i> < 0.05. n.s. indicates non-significance with <i>P</i> > 0.05. The responses at the ERG b_(max,rod) light intensity, indicated by the “†”, were statistically evaluated and are represented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0159776#pone.0159776.s001" target="_blank">S1A Fig</a>.</p

Summary of OCT findings.

<p>Summary of OCT findings.</p

Flibanserin increases anti-apoptotic and antioxidant gene expression.

<p><b>(A)</b> RT-qPCR shows that 48 hours following light-exposure, expression of <i>Creb</i>, <i>Bcl-2</i>, <i>Cast1</i>, <i>Sod1</i>, and <i>Cat</i> are significantly increased in flibanserin-injected mice versus vehicle-injected mice. <b>(B)</b> After 72 hours, expression of <i>Creb</i>, <i>c-Jun</i>, <i>c-Fos</i>, <i>Bcl-2</i>, <i>Cast1</i>, <i>Nqo1</i>, and <i>Sod1</i> are significantly increased in flibanserin-injected mice versus vehicle-injected mice. Analysis was performed using the ΔΔCt method with β-actin as the internal control. Significance was determined using a multiple t-test analysis (* indicates <i>P</i> < 0.05). cAMP Response Binding-element Protein (<i>Creb</i>), Cyclin D1 (<i>Cd1</i>), B-cell lymphoma 2 (<i>Bcl-2</i>), Calpastatin (<i>Cast1</i>), Nitric Oxide Synthase (<i>Nos1</i>), NAD(P)H quinone dehydrogenase 1 (<i>Nqo1</i>), Superoxide Dismutase 1 (<i>Sod1</i>), Catalase (<i>Cat</i>), Metallothionein 1 (<i>Mt1</i>).</p

A 5-day time course of flibanserin protects retinal morphology from bright light exposure in a dose-dependent manner.

<p>(<b>A</b>) Representative linear SD-OCT scans of nasal retina show increasing outer nuclear layer thickness with flibanserin treatment, in a dose-dependent manner. A naïve non-light damaged mouse shows an easily distinguishable OPL, ONL, OLM, IS/OS, and RPE. GCL-ganglion cell layer, IPL-inner plexiform layer, INL-inner nuclear layer, OPL-outer plexiform layer, ONL-outer nuclear layer, OLM-outer limiting membrane, IS/OS-inner segment/outer segment junction, RPE-retinal pigment epithelium. (<b>B i</b>) A spider graph of average right-eye receptor plus values demonstrates that flibanserin-mediated protection increases in a dose-dependent manner starting at 3 mg/kg, with a 15 mg/kg dose showing comparable receptor plus thicknesses to naïve (non-light damaged) mice. The gray area indicates ± 2 SD of the naïve averaged data. (<b>B ii, iii</b>) Receptor plus thicknesses, with each dot representing average right and left eye thickness from one mouse, demonstrate that 15 mg/kg flibanserin achieves complete morphological protection in the <b>(B ii)</b> temporal quadrant and the <b>(B iii)</b> nasal quadrant. Group averages are represented as mean ± standard error bar. * indicates a significant difference from both the vehicle-treated group and the naïve group, <i>P</i> < 0.05. n.s. indicates non-significance with <i>P</i> > 0.05.</p