Retinal gene therapy is increasingly recognised as a novel molecular intervention that has huge potential in treating common causes of blindness, the majority of which have a genetic aetiology. 1-5 Choroideremia is a chronic X-linked retinal degeneration that was first described in 1872. 6 It leads to progressive blindness due to deficiency of Rab-escort protein 1 (REP1). We designed an adenoassociated viral vector to express REP1 and assessed it in a gene therapy clinical trial by subretinal injection in 14 patients with choroideremia. The primary endpoint was vision change in treated eyes two years after surgery compared to unoperated fellow eyes. Despite complications in two patients, visual acuity improved in the 14 treated eyes over controls (median 4.5 letter gain, vs 1.5 letter loss, p=0.04), with six treated eyes gaining more than one line of vision (>5 letters). The results suggest that retinal gene therapy can sustain and improve visual acuity in a cohort of predominantly late stage choroideremia patients in whom rapid visual acuity loss would ordinarily be predicted.

Purpose To analyse the spectrum of bacterial keratitis isolates and their in vitro antibiotic susceptibilities over a 10-year period in Oxford, UK; and to compare the in vitro efficacy of ciprofloxacin with that of the combination of gentamicin and cefuroxime over the same period. Methods All culture-positive corneal scrapes received from the Oxford Eye Hospital between July 1999 and June 2009 were identified retrospectively using a local microbiology database. For analysis of trends over time, the data was split into two equal 5-year periods. Statistical analysis was done using the v 2 and Fisher exact tests. Results Over the 10-year study period, 467 corneal scrapes were performed of which 252 (54.0%) had positive bacterial cultures, growing a total of 267 organisms. The most commonly isolated bacteria were Staphylococci (40.1%) followed by Pseudomonas species (28.5%), other Gramnegative species (17.2%), Streptococci (7.1%), and Corynebacteria (6.0%). Between the first and second time periods there was an increase in the number of coagulase-negative Staphylococci and an increased resistance of the non-Pseudomonas Gram-negative group to chloramphenicol. Of the 189 isolates tested for sensitivity to both empirical antibiotic regimens, 176 (93.2%) were susceptible to ciprofloxacin whereas 188 (99.5%) were susceptible to either gentamicin or cefuroxime (P ¼ 0.0015). Conclusions The spectrum of bacterial keratitis isolates and their in vitro antibiotic sensitivity patterns have generally remained stable over time. The combination of gentamicin and cefuroxime provides a broader spectrum of antimicrobial cover than ciprofloxacin monotherapy in Oxford, although both regimens continue to be appropriate choices for the initial management of this condition.

Ocular angiogenesis and macular oedema are major causes of sight loss across the world. Aberrant neovascularisation, which may arise secondary to numerous disease processes, can result in reduced vision as a result of oedema, haemorrhage, and scarring. The development of antivascular endothelial growth factor (anti-VEGF) agents has revolutionised the treatment of retinal vasogenic conditions. These drugs are now commonly employed for the treatment of a plethora of ocular pathologies including choroidal neovascularisation, diabetic macular oedema, and retinal vein occlusion to name a few. In this paper, we will explore the current use of anti-VEGF in a variety of retinal diseases and the impact that these medications have had on visual outcome for patients.

Over the time period of this study there was no evidence of emerging resistance to empirical antibiotics which are commonly used for the treatment of bacterial endophthalmitis. Infection with coagulase-negative Staphylococci was associated with a good visual outcome, whilst infection with Streptococcus spp. or Haemophilus influenzae was associated with a poor visual outcome.

The woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) has been included in the transgene cassette of adeno-associated virus (AAV) in several gene therapy clinical trials, including those for inherited retinal diseases. However, the extent to which WPRE increases transgene expression in the retina is still unclear. To address this question, AAV2 vectors containing a reporter gene with and without WPRE were initially compared in vitro and subsequently in vivo by subretinal delivery in mice. In both instances, the presence of WPRE led to significantly higher levels of transgene expression as measured by fundus fluorescence, western blot, and immunohistochemistry. The two vectors were further compared in human retinal explants derived from patients undergoing clinically indicated retinectomy, where again the presence of WPRE resulted in an enhancement of reporter gene expression. Finally, an analogous approach using a transgene currently employed in a clinical trial for choroideremia delivered similar results both in vitro and in vivo, confirming that the WPRE effect is transgene independent. Our data fully support the inclusion of WPRE in ongoing and future AAV retinal gene therapy trials, where it may allow a therapeutic effect to be achieved at an overall lower dose of vector.

Recombinant adeno-associated viral (AAV) vectors have been successfully employed as the mode of gene delivery in several clinical trials for the treatment of inherited retinal diseases to date. The design of such vectors is critical in determining cellular tropism and level of subsequent gene expression that may be achieved following viral delivery. Here we describe a system for living retinal tissue extraction, ex vivo culture, viral transduction and assessment of transgene expression that may be used to assess viral constructs for gene therapy in the human retina at a preclinical stage.

Rhodopsin (RHO) gene mutations are a common cause of autosomal dominant retinitis pigmentosa (ADRP). The need to suppress toxic protein expression together with mutational heterogeneity pose challenges for treatment development. Mirtrons are atypical RNA interference effectors that are spliced from transcripts as short introns. Here, we develop a novel mirtron-based knockdown/replacement gene therapy for the mutation-independent treatment of RHO-related ADRP, and demonstrate efficacy in a relevant mammalian model. Splicing and potency of rhodopsin-targeting candidate mirtrons are initially determined, and a mirtron-resistant codon-modified version of the rhodopsin coding sequence is validated in vitro. These elements are then combined within a single adeno-associated virus (AAV) and delivered subretinally in a RhoP23H knock-in mouse model of ADRP. This results in significant mouse-to-human rhodopsin RNA replacement and is associated with a slowing of retinal degeneration. This provides proof of principle that synthetic mirtrons delivered by AAV are capable of reducing disease severity in vivo.

PurposeTreatment of inherited retinal degenerations using adeno-associated viral (AAV) vectors involves delivery by subretinal injection. In the latter stages, alteration of normal anatomy may cause difficulty in visualizing the retinotomy, retinal detachment extension, and vector diffusion. Vital dyes may be useful surgical adjuncts, but their safety and impact on AAV transduction are largely unknown.MethodsThe effects of Sodium Fluorescein (SF), Membrane Blue (MB), and Membrane Blue Dual (DB) at a range of dilutions were assessed on human embryonic kidney cells in vitro using an AAV2-green fluorescent protein (GFP) reporter at different multiplicities of infection. Flow cytometry analysis was performed to assess both cell viability and transduction efficiency. The effect on quantitative (q)PCR titer was determined. Balanced salt solution (BSS) or dilute DB (1:5 in BSS) were delivered subretinally into left/right eyes of C57BL/6J mice (n = 12). Retinal structure and function were analyzed by optical coherence tomography, autofluorescence, dark-and light-adapted full-field electroretinography.ResultsDB and MB were not toxic at any concentration tested, SF only when undiluted. The presence of dyes did not adversely affect the genomic titer. DB even increased the values, due to presence of surfactant in the formulation. AAV2-GFP transduction efficiency was not reduced by the dyes. No structural and functional toxic effects were observed following subretinal delivery of DB.ConclusionsOnly undiluted SF affected cell viability. No effects on qPCR titer and transduction efficiency were observed. DB does not appear toxic when delivered subretinally and improves titer accuracy. DB may therefore be a safe and helpful adjunct during gene therapy surgery.Translational RelevanceThis paper might be of interest to the retinal gene therapy community: it is a “bench to bedside” research paper about the potential use of dyes as a surgical adjunct during the gene therapy surgery. We have tested the potential toxicity and impact on transduction efficiency in an in vitro and in vivo model.