Jasmina Cehajic-Kapetanovic

Restoration of Vision with Ectopic Expression of Human Rod Opsin

Summary Many retinal dystrophies result in photoreceptor loss, but the inner retinal neurons can survive, making them potentially amenable to emerging optogenetic therapies. Here, we show that ectopically expressed human rod opsin, driven by either a non-selective or ON-bipolar cell-specific promoter, can function outside native photoreceptors and restore visual function in a mouse model of advanced retinal degeneration. Electrophysiological recordings from retinal explants and the visual thalamus revealed changes in firing (increases and decreases) induced by simple light pulses, luminance increases, and naturalistic movies in treated mice. These responses could be elicited at light intensities within the physiological range and substantially below those required by other optogenetic strategies. Mice with rod opsin expression driven by the ON-bipolar specific promoter displayed behavioral responses to increases in luminance, flicker, coarse spatial patterns, and elements of a natural movie at levels of contrast and illuminance (≈50–100 lux) typical of natural indoor environments. These data reveal that virally mediated ectopic expression of human rod opsin can restore vision under natural viewing conditions and at moderate light intensities. Given the inherent advantages in employing a human protein, the simplicity of this intervention, and the quality of vision restored, we suggest that rod opsin merits consideration as an optogenetic actuator for treating patients with advanced retinal degeneration.

Chemogenetic Activation of Melanopsin Retinal Ganglion Cells Induces Signatures of Arousal and/or Anxiety in Mice

Summary Functional imaging and psychometric assessments indicate that bright light can enhance mood, attention, and cognitive performance in humans. Indirect evidence links these events to light detection by intrinsically photosensitive melanopsin-expressing retinal ganglion cells (mRGCs) [1, 2, 3, 4, 5, 6, 7, 8, 9]. However, there is currently no direct demonstration that mRGCs can have such an immediate effect on mood or behavioral state in any species. We addressed this deficit by using chemogenetics to selectively activate mRGCs, simulating the excitatory effects of bright light on this cell type in dark-housed mice. This specific manipulation evoked circadian phase resetting and pupil constriction (known consequences of mRGC activation). It also induced c-Fos (a marker of neuronal activation) in multiple nuclei in the hypothalamus (paraventricular, dorsomedial, and lateral hypothalamus), thalamus (paraventricular and centromedian thalamus), and limbic system (amygdala and nucleus accumbens). These regions influence numerous aspects of autonomic and neuroendocrine activity and are typically active during periods of wakefulness or arousal. By contrast, c-Fos was absent from the ventrolateral preoptic area (active during sleep). In standard behavioral tests (open field and elevated plus maze), mRGC activation induced behaviors commonly interpreted as anxiety like or as signs of increased alertness. Similar changes in behavior could be induced by bright light in wild-type and rodless and coneless mice, but not melanopsin knockout mice. These data demonstrate that mRGCs drive a light-dependent switch in behavioral motivation toward a more alert, risk-averse state. They also highlight the ability of this small fraction of retinal ganglion cells to realign activity in brain regions defining widespread aspects of physiology and behavior.

Augmentin duo? in the treatment of childhood blepharokeratoconjunctivitis.

PURPOSE To report the use of Augmentin Duo 400/57 (GlaxoSmithKline, Middlesex, UK) in the treatment of childhood blepharokeratoconjunctivitis (BKC). METHODS This is a retrospective interventional case series. The case notes of 7 consecutive patients treated with Augmentin Duo 400/57 for BKC during 18 months were reviewed. Diagnostic criteria for BKC were blepharitis including recurrent chalazia and meibomian gland dysfunction, eyelid margin telangiectasia and facial rosacea, recurrent episodes of chronic red eye, photophobia, watering, punctate superficial keratopathy, corneal neovascularization, and corneal ulcers. RESULTS Seven children (age range: 6 to 14 years) were diagnosed as having BKC. All children received systemic Augmentin Duo 400/57 and showed considerable improvement within the first month of therapy. Six children had no recurrences during a mean follow-up of 6 months. No patients experienced any side effects from this treatment. CONCLUSIONS Augmentin Duo 400/57 has not previously been reported in the treatment of BKC in children. In this case series, Augmentin Duo 400/57 proved to be at least as effective as current treatments with systemic erythromycin or doxycycline with the advantage of a twice-daily dosage and a superior side-effect profile.

Chemogenetic Activation of ipRGCs Drives Changes in Dark-Adapted (Scotopic) Electroretinogram.

Purpose The purpose of this study was to investigate the impact of activating melanopsin-expressing intrinsically photosensitive retinal ganglion cells (ipRGCs) on dark-adapted (scotopic) electroretinograms (ERG). Methods We used mice (Opn4Cre/+) expressing cre recombinase in melanopsin-expressing cells for a targeted gene delivery of a chemogenetic Gq-coupled receptor, hM3Dq, to ipRGCs. Intraperitoneal injection of clozapine N-oxide (CNO) at 5 mg/kg was used for acute activation of hM3Dq and thus excitation of ipRGCs in darkness. Dark-adapted flash ERGs were recorded across a 9-fold range of irradiances from hM3Dq Opn4Cre/+ and control Opn4Cre/+ mice before and after intraperitoneal injection of CNO. A- and b-wave amplitudes and implicit times and oscillatory potentials (OPs) were analyzed. Paired-flash stimuli were used to isolate cone-driven responses. Results Clozapine N-oxide application suppressed a- and b-wave amplitudes of the dark-adapted ERG across the flash intensity range in hM3Dq Opn4Cre/+ mice compared to control mice. Examination of the normalized irradiance-response functions revealed a shift in b-wave but not a-wave sensitivity. No changes in a- and b-wave implicit times were detected. Total OP amplitudes were also reduced in hM3Dq Opn4Cre/+ mice compared to controls following CNO administration. The paired-flash method revealed reduction in both the first (rods and cones) and second (cones only) flash response. Conclusions Acute and selective activation of ipRGCs modulates the amplitude of both a- and b-waves of the scotopic ERG, indicating that the influence of this ganglion cell class on the retinal physiology extends to the photoreceptors as well as their downstream pathways.

Efficacy and Safety of Glycosidic Enzymes for Improved Gene Delivery to the Retina following Intravitreal Injection in Mice

Viral gene delivery is showing great promise for treating retinal disease. Although subretinal vector delivery has mainly been used to date, intravitreal delivery has potential advantages if low retinal transduction efficiency can be overcome. To this end, we investigated the effects of co-injection of glycosaminoglycan-degrading enzymes, singly or in combination, with AAV2 as a method of increasing retinal transduction. Experiments using healthy mice demonstrated that these enzymes enhance retinal transduction. We found that heparinase III produced the greatest individual effect, and this was enhanced further by combination with hyaluronan lyase. In addition, this optimized AAV2-enzyme combination led to a marked improvement in transduction in retinas with advanced retinal degeneration compared with AAV2 alone. Safety studies measuring retinal function by flash electroretinography indicated that retinal function was unaffected in the acute period and at least 12 months after enzyme treatment, whereas pupillometry confirmed that retinal ganglion cell activity was unaffected. Retinal morphology was not altered by the enzyme injection. Collectively these data confirm the efficacy and safety of this intravitreal approach in enhancing retinal transduction efficiency by AAV in rodents. Translating this method into other species, such as non-human primates, or for clinical applications will have challenges and require further studies.

Enhancement of light sensitivity in retinal degeneration in mice by use of novel optogenetic approaches

Abstract Background Inherited retinal degenerations (including retinitis pigmentosa) that lead to irreversible blindness due to progressive loss of rods and cones in the outer retina affect one in 2500 people worldwide. Despite this high prevalence, there is no cure. The therapeutic approaches that are being explored in clinical trials have low efficacy and high complication rates (gene augmentation) or low resolution (artificial retinal implants). In this study we investigated the effect of ectopic expression of two light sensitive proteins, human melanopsin and human rhodopsin, on visual function in a mouse model (rd1) of advanced retinal degeneration using enhanced gene therapy. We hypothesised that this combined gene therapy and optogenetic approach might render surviving inner retinal cells photosensitive and enhance visual function. Methods The melanopsin or rhodopsin expressing adeno-associated virus serotype 2 (AAV2) vector was injected intravitreally into the eyes of adult rd1 mice with a combination of glycosidic enzymes, which we have previously shown to improve retinal transduction efficiency. Mouse visual function was assessed with pupillometry at 6 weeks post treatment. The downstream effects of more complex visual processing were examined by in-vivo electrophysiology recordings from a mouse lateral geniculate nucleus (LGN), a primary input for retinal ganglion cells. Findings We found expression of ectopic human melanopsin in a variety of surviving retinal cells in rd/rd mouse eyes treated with melanopsin, including ganglion and bipolar cells. The pupillary light reflex showed an enhancement in visual sensitivity, by two orders of magnitude, in eyes treated with melanopsin. We also found that rhodopsin treatment led to a similar shift in sensitivity. Interpretation Melanopsin and rhodopsin, expressed in degenerating retina, have potential to enhance light sensitivity. We are now assessing the benefits of this enhanced sensitivity in terms of changes in visual responses in mouse LGN. Both receptors operate within physiological light conditions, unlike present optogenetic approaches (such as microbial channel rhodopsin-2 [Ch2]), which require stimulation with very high light intensities that are potentially toxic to the retina. Furthermore, both melanopsin and rhodopsin are native mammalian proteins with minimum risk of adverse immune responses compared with Ch2, making them preferred candidates for use in future human trials. Funding UK Medical Research Council.

Outer retinal transduction can be achieved after intravitreal delivery of adeno-associated virus serotype 2 vector with glycosidic enzymes

Abstract Background Gene therapies for retinal disorders, including in current clinical trials, so far have relied on subretinal delivery of adeno-associated virus (AAV) vectors carrying therapeutic DNA into outer retinal cells. Subretinal injection has many limitations over the less-invasive route of administration into the vitreal cavity. However, at present only limited retinal transduction can be achieved after intravitreal delivery of AAV vectors. We hypothesise that the inner limiting membrane and extracellular matrix proteoglycans act as a barrier to AAV vector entry into and movement across the retina. Therefore, glycosidic enzymes, which degrade these extracellular barriers, can improve retinal gene therapy. In this study we investigated the effects of enzymatic digestion of extracellular matrices on the depth of vector penetration into the retina. Methods The green fluorescent protein (GFP)-expressing AAV serotype 2 (AAV2) vector was co-injected intravitreally with glycosidic enzymes at their optimum concentration. Efficacy of virus transduction was assessed by visualising fluorescence in histological cross-sections with fluorescence microscopy. We also analysed safety of these treatments and retinal function using electroretinography. Findings Glycosidic enzymes led to a significant improvement in retinal transduction after intravitreal delivery of AAV2. These enzymes markedly improved transduction of the outer retina, including photoreceptor cells. Electroretinograms were unchanged (compared with controls) even at much higher doses of enzymes than were needed for optimum retinal transduction. Interpretation AAV2-mediated retinal transduction is improved by co-injection of glycosidic enzymes into the vitreous. Improved transduction efficiency may allow intravitreal injection to become the preferred route for delivering gene therapy to the inner and outer retina in both preclinical and clinical settings. Funding UK Medical Research Council.