Summary Background Safety and efficacy have been shown in a phase 1 dose-escalation study involving a unilateral subretinal injection of a recombinant adeno-associated virus (AAV) vector containing the RPE65 gene (AAV2-hRPE65v2) in individuals with inherited retinal dystrophy caused by RPE65 mutations. This finding, along with the bilateral nature of the disease and intended use in treatment, prompted us to determine the safety of administration of AAV2-hRPE65v2 to the contralateral eye in patients enrolled in the phase 1 study. Methods In this follow-on phase 1 trial, one dose of AAV2-hRPE65v2 (1·5 × 1011 vector genomes) in a total volume of 300 μL was subretinally injected into the contralateral, previously uninjected, eyes of 11 children and adults (aged 11–46 years at second administration) with inherited retinal dystrophy caused by RPE65 mutations, 1·71–4·58 years after the initial subretinal injection. We assessed safety, immune response, retinal and visual function, functional vision, and activation of the visual cortex from baseline until 3 year follow-up, with observations ongoing. This study is registered with ClinicalTrials.gov, number NCT01208389. Findings No adverse events related to the AAV were reported, and those related to the procedure were mostly mild (dellen formation in three patients and cataracts in two). One patient developed bacterial endophthalmitis and was excluded from analyses. We noted improvements in efficacy outcomes in most patients without significant immunogenicity. Compared with baseline, pooled analysis of ten participants showed improvements in mean mobility and full-field light sensitivity in the injected eye by day 30 that persisted to year 3 (mobility p=0·0003, white light full-field sensitivity p<0·0001), but no significant change was seen in the previously injected eyes over the same time period (mobility p=0·7398, white light full-field sensitivity p=0·6709). Changes in visual acuity from baseline to year 3 were not significant in pooled analysis in the second eyes or the previously injected eyes (p>0·49 for all time-points compared with baseline). Interpretation To our knowledge, AAV2-hRPE65v2 is the first successful gene therapy administered to the contralateral eye. The results highlight the use of several outcome measures and help to delineate the variables that contribute to maximal benefit from gene augmentation therapy in this disease. Funding Center for Cellular and Molecular Therapeutics at The Children’s Hospital of Philadelphia, Spark Therapeutics, US National Institutes of Health, Foundation Fighting Blindness, Institute for Translational Medicine and Therapeutics, Research to Prevent Blindness, Center for Advanced Retinal and Ocular Therapeutics, Mackall Foundation Trust, F M Kirby Foundation, and The Research Foundation—Flanders.

The acute hippocampal slice preparation is a convenient, in vitro model widely used to study the biological basis of synaptic plasticity. Although slices may preserve their electrophysiological properties for several hours, profound molecular changes in response to the injury caused by the slicing procedure are likely to occur. To determine the magnitude and duration of these changes we examined the post-slicing expression kinetics of three classes of genes known to be implicated in long-term synaptic plasticity: glutamate AMPA receptors (GluR), transcription factors and neurotrophins. Slicing resulted in a striking loss of GluR1 and GluR3, but not of GluR2 proteins suggesting that rapid changes in the composition of major neurotransmitter receptors may occur. Slicing caused a significant induction of the transcription factors c-fos, zif268, CCAAT enhancer binding protein (C/EBP) b and d mRNAs and of the neurotrophin brain-derived neurothophic factor (BDNF) mRNA. In contrast, there was no augmentation, and sometimes a decline, in the levels of the corresponding proteins. These data reveal that significant discrepancies exist between the slice preparation and the intact hippocampus in terms of the metabolism of molecular components known to be involved in synaptic plasticity.

Purpose To quantify the amount of drug loss from cadaveric human eyes which are injected via the pars plana with a known volume of dye at variable intraocular pressures. Methods Eight cadaver eyes were divided into two intraocular pressure groups: normal (15 mmHg, 4 eyes) or high (30 mmHg, 4 eyes). Each eye was injected with 50 µl of hematoxylin dye and the subsequent reflux was immediately collected on a Schirmers test strip. The test strip was scanned and digitally analyzed to determine the area of saturation and total color intensity present. Using a previously established equation, total volume of reflux and amount of dye within that reflux were calculated. Results The average total volume of refluxed fluid was 1.68 µL (median: 0.62 µL), with a range of 0 µL to 8.05 µL. The average volume of refluxed dye was 0.37 µL (median: 0.08 µL), with a range of 0 µL to 2.15 µL. On average only 0.74% of the original 50 µL of injected dye was lost (median: 0.15%), with a range from 0% to 4.30%. Conclusion Although the presence of subconjunctival bleb formation after intravitreal injection may be concerning to the clinician, our data shows that only a very small amount of the injected therapeutic agent is lost in the reflux.

Background Reflux following intravitreal injection is a common phenomenon, but it is unknown how much, if any, medication is lost as a result. Reflux is known to be a combination of vitreous and the injected agent, but the relative composition is unknown. This paper describes a novel method for the measurement of the volume and composition of reflux and presents data from porcine eyes. Methods Twenty porcine eyes were injected with 0.05 ml of dye at intraocular pressures (IOPs) of 15, 20, 25 and 30 mmHg (5 eyes per subgroup). Reflux was captured on filter paper and the area of saturation and color intensity of the dye were digitally analyzed. Total refluxed volume and proportion of dye vs. vitreous fluid were calculated from linear regression lines created from known standards. Results Average (median) total volume of reflux from all eyes was 1.19 μL (0.93 μL), volume of injected dye refluxed was 0.47 μL (0.11 μL), and composition of reflux was 20.8% dye (15.5%). Less than 1% of the injected dye was lost to reflux. There were no differences between IOP groups in the total volume refluxed, the total amount of dye refluxed, the average composition of the reflux, or the amount of injected dye refluxed (df=3 for all comparisons; p=0.58, p=0.51, p=0.55, p=0.51, respectively). Conclusions This novel method allows for measurement of quantity and composition of reflux following intravitreal injection in vitro. While reflux occurs frequently, it is predominantly composed of vitreous, not the injected agent. In fact, less than one percent of the original injection was lost to reflux.

PURPOSE To develop a rabbit model for continuous curvilinear capsulorhexis (CCC) instruction. SETTING University of California San Francisco, San Francisco, California, USA. DESIGN Experimental study. METHODS Isolated rabbit lenses were immersed in 2% to 8% paraformaldehyde (PFA) fixative from 15 minutes to 6 hours. Rabbit eyes were treated by substituting aqueous with 2% to 4% PFA for 30 minutes to 6 hours, followed by washes with a balanced salt solution. Treated lenses and eyes were held in purpose-designed holders using vacuum. A panel of 6 cataract surgeons with 5 to 15 years of experience performed CCC on treated lenses and eyes and responded to a questionnaire regarding the utility of these models for resident teaching using a 5-item Likert scale. RESULTS The expert panel found that rabbit lenses treated with increasing amounts of fixative simulated CCC on human lens capsules from the third to the seventh decade of life. The panel also found fixative-treated rabbit eyes to simulate some of the experience of CCC within the human anterior chamber but noted a shallower anterior chamber depth, variation in pupil size, and corneal clouding under some treatment conditions. CONCLUSIONS Experienced cataract surgeons who performed CCC on these rabbit models strongly agreed that isolated rabbit lenses treated with fixative provide a realistic simulation of CCC in human patients and that both models were useful tools for capsulorhexis instruction. Results indicate that rabbit lenses treated with 8% PFA for 15 minutes is a model with good fidelity for CCC training.