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

A one-day time course of flibanserin can preserve retinal morphology and function.

<p><b>(A i)</b> A spider graph representing average right-eye receptor plus values demonstrates that a single dose of 6 mg/kg flibanserin or greater can significantly preserve retinal morphology to thicknesses comparable to naïve mice. The gray area indicates ± 2 SD of the naïve averaged data. (<b>A ii, iii</b>) Receptor plus thicknesses, with each dot representing average right and left eye thickness from one mouse, demonstrate that 6 mg/kg flibanserin provides morphological protection in the <b>(A ii)</b> temporal quadrant and the <b>(A iii)</b> nasal quadrant. Group averages are represented as mean ± standard error bar. <b>(B)</b> Mice treated with doses of 6 mg/kg and greater of flibanserin showed significantly higher ERG b-wave and a-wave responses at the highest flash intensity (3.55 log cd•s/m²) as compared to vehicle-treated mice. “†” indicates the greatest flash intensity that elicits a rod-only response (b_(max,rod)). Group differences at the “†” flash intensity 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">S1B Fig</a>. Individual ERGs were averaged with ERGs for mice within 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.</p

Summary of OCT findings.

<p>Summary of OCT findings.</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

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

Summary of ERG parameters.

<p>Summary of ERG parameters.</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

Targeting Photoreceptors via Intravitreal Delivery Using Novel, Capsid-Mutated AAV Vectors

<div><p>Development of viral vectors capable of transducing photoreceptors by less invasive methods than subretinal injection would provide a major advancement in retinal gene therapy. We sought to develop novel AAV vectors optimized for photoreceptor transduction following intravitreal delivery and to develop methodology for quantifying this transduction <i>in vivo</i>. Surface exposed tyrosine (Y) and threonine (T) residues on the capsids of AAV2, AAV5 and AAV8 were changed to phenylalanine (F) and valine (V), respectively. Transduction efficiencies of self-complimentary, capsid-mutant and unmodified AAV vectors containing the smCBA promoter and mCherry cDNA were initially scored <i>in vitro</i> using a cone photoreceptor cell line. Capsid mutants exhibiting the highest transduction efficiencies relative to unmodified vectors were then injected intravitreally into transgenic mice constitutively expressing a Rhodopsin-GFP fusion protein in rod photoreceptors (Rho-GFP mice). Photoreceptor transduction was quantified by fluorescent activated cell sorting (FACS) by counting cells positive for both GFP and mCherry. To explore the utility of the capsid mutants, standard, (non-self-complementary) AAV vectors containing the human rhodopsin kinase promoter (hGRK1) were made. Vectors were intravitreally injected in wildtype mice to assess whether efficient expression exclusive to photoreceptors was achievable. To restrict off-target expression in cells of the inner and middle retina, subsequent vectors incorporated multiple target sequences for miR181, an miRNA endogenously expressed in the inner and middle retina. Results showed that AAV2 containing four Y to F mutations combined with a single T to V mutation (quadY−F+T−V) transduced photoreceptors most efficiently. Robust photoreceptor expression was mediated by AAV2(quadY−F+T−V) −hGRK1−GFP. Observed off-target expression was reduced by incorporating target sequence for a miRNA highly expressed in inner/middle retina, miR181c. Thus we have identified a novel AAV vector capable of transducing photoreceptors following intravitreal delivery to mouse. Furthermore, we describe a robust methodology for quantifying photoreceptor transduction from intravitreally delivered AAV vectors.</p></div

<i>In vivo</i> analysis of AAV2-based vectors containing the hGRK1 promoter.

<p>Fundus images paired with immunohistochemistry of frozen retinal cross-sections from C57BL/6 mice taken 4 weeks post injection with AAV2, AAV2(quad Y-F), and AAV2(quad Y-F +T-V) vectors containing hGRK1-GFP (7.5×10⁹ vg delivered). Identical gain and exposures were used for fundoscopy. All tissue sections were imaged at 20X, with identical gain and exposure settings. GFP expression is shown in green. Nuclei were counterstained with DAPI (blue). RPE- retinal pigment epithelium, IS/OS- inner segments/outer segments, ONL- outer nuclear layer, INL- inner nuclear layer, GCL- ganglion cell layer.</p

Qualitative comparison of unmodified and capsid mutated AAV vectors <i>in vivo</i><b>.</b>

<p>Fundoscopy (red channel only) of Rho-GFP mice 4 weeks post-injection with unmodified and capsid-mutated scAAV-smCBA-mCherry vectors (1.5×10⁹ vg delivered). Exposure and gain settings were the same across all images.</p