The in vivo pattern of AID targeting to immunoglobulin switch regions deduced from mutation spectra in msh2−/− ung−/− mice

Immunoglobulin (Ig) class switching is initiated by deamination of C→U within the immunoglobulin heavy chain locus, catalyzed by activation-induced deaminase (AID). In the absence of uracil-DNA glycosylase (UNG) and the homologue of bacterial MutS (MSH)–2 mismatch recognition protein, the resultant U:G lesions are not processed into switching events but are fixed by replication allowing sites of AID-catalyzed deamination to be identified by the resulting C→T mutations. We find that AID targets cytosines in both donor and acceptor switch regions (S regions) with the deamination domains initiating ∼150 nucleotides 3′ of the I exon start sites and extending over several kilobases (the IgH intronic enhancer is spared). Culturing B cells with interleukin 4 or interferon γ specifically enhanced deamination around Sγ1 and Sγ2a, respectively. Mutation spectra suggest that, in the absence of UNG and MSH2, AID may occasionally act at the μ switch region in an apparently processive manner, but there is no marked preference for targeting of the transcribed versus nontranscribed strand (even in areas capable of R loop formation). The data are consistent with switch recombination being triggered by transcription-associated, strand-symmetric AID-mediated deamination at both donor and acceptor S regions with cytokines directing isotype specificity by potentiating AID recruitment to the relevant acceptor S region

Enhancement of Adeno-Associated Virus-Mediated Gene Therapy Using Hydroxychloroquine in Murine and Human Tissues.

The therapeutic effects of gene therapy using adeno-associated virus (AAV) vectors are dependent on the efficacy of viral transduction. Currently, we have reached the safe limits of AAV vector dose, beyond which damaging inflammatory responses are seen. To improve the efficacy of AAV transduction, we treated mouse embryonic fibroblasts, primate retinal pigment epithelial cells, and human retinal explants with hydroxychloroquine (HCQ) 1 h prior to transduction with an AAV2 vector encoding GFP driven by a ubiquitous CAG promoter. This led to a consistent increase in GFP expression, up to 3-fold, compared with vector alone. Comparing subretinal injections of AAV2.CAG.GFP vector alone versus co-injection with 18.75 μM HCQ in paired eyes in mice, mean GFP expression was 4.6-fold higher in retinae co-treated with HCQ without retinal toxicity. A comparative 5.9-fold effect was seen with an AAV8(Y733F).GRK1.GFP vector containing the photoreceptor-specific rhodopsin kinase promoter. While the mechanism of action remains to be fully elucidated, our data suggest that a single pulse of adjunctive HCQ could safely improve AAV transduction in vivo, thus providing a novel strategy for enhancing the clinical effects of gene therapy

Interactions between apolipoprotein E metabolism and retinal inflammation in age-related macular degeneration

Age-related macular degeneration (AMD) is a multifactorial retinal disorder that is a major global cause of severe visual impairment. The development of an effective therapy to treat geographic atrophy, the predominant form of AMD, remains elusive due to the incomplete understanding of its pathogenesis. Central to AMD diagnosis and pathology are the hallmark lipid and proteinaceous deposits, drusen and reticular pseudodrusen, that accumulate in the subretinal pigment epithelium and subretinal spaces, respectively. Age-related changes and environmental stressors, such as smoking and a high-fat diet, are believed to interact with the many genetic risk variants that have been identified in several major biochemical pathways, including lipoprotein metabolism and the complement system. The APOE gene, encoding apolipoprotein E (APOE), is a major genetic risk factor for AMD, with the APOE2 allele conferring increased risk and APOE4 conferring reduced risk, in comparison to the wildtype APOE3. Paradoxically, APOE4 is the main genetic risk factor in Alzheimer’s disease, a disease with features of neuroinflammation and amyloid-beta deposition in common with AMD. The potential interactions of APOE with the complement system and amyloid-beta are discussed here to shed light on their roles in AMD pathogenesis, including in drusen biogenesis, immune cell activation and recruitment, and retinal inflammation

A Review of the Landscape of Targeted Immunomodulatory Therapies for Non-Infectious Uveitis

<p><b>Article full text</b></p> <p><br></p> <p>The full text of this article can be found here<b>.</b> <a href="https://link.springer.com/article/10.1007/s40123-017-0115-5">https://link.springer.com/article/10.1007/s40123-017-0115-5</a></p><p></p> <p><br></p> <p><b>Provide enhanced content for this article</b></p> <p><br></p> <p>If you are an author of this publication and would like to provide additional enhanced content for your article then please contact <a href="http://www.medengine.com/Redeem/”mailto:[email protected]”"><b>[email protected]</b></a>.</p> <p><br></p> <p>The journal offers a range of additional features designed to increase visibility and readership. All features will be thoroughly peer reviewed to ensure the content is of the highest scientific standard and all features are marked as ‘peer reviewed’ to ensure readers are aware that the content has been reviewed to the same level as the articles they are being presented alongside. Moreover, all sponsorship and disclosure information is included to provide complete transparency and adherence to good publication practices. This ensures that however the content is reached the reader has a full understanding of its origin. No fees are charged for hosting additional open access content.</p> <p><br></p> <p>Other enhanced features include, but are not limited to:</p> <p><br></p> <p>• Slide decks</p> <p>• Videos and animations</p> <p>• Audio abstracts</p> <p>• Audio slides</p

Correlation of optical coherence tomography and autofluorescence in the outer retina and choroid of patients with choroideremia

PURPOSE. To evaluate the relationships between RPE, photoreceptor, and choroidal degeneration in choroideremia. METHODS. Enhanced-depth imaging optical coherence tomography (EDI-OCT), scanning laser ophthalmoscopy (SLO), and autofluorescence (AF) were performed on 39 patients (78 eyes) with choroideremia. The edges of surviving outer retina on OCTand residual AF were aligned. The distribution of outer retinal tubulations was mapped over a range of ages (16–71 years), and comparison made between pre- and postsubretinal gene therapy. Subfoveal choroidal thickness (SFCT) was compared between 23 choroideremia patients (42 eyes) and 20 age- and refraction-matched male controls (40 eyes). RESULTS. The edges of RPE AF aligned with a reduction in outer nuclear layer thickness (Spearman’s rho = 0.9992). Correlation was also found between the quality of AF and integrity of ellipsoid zone within islands of surviving retina. Tubulations existed in 71 of 78 (91%) eyes with choroideremia and remained stable following gene therapy. Subfoveal choroidal thickness was reduced at baseline in choroideremia (179.7 ± 17.2 μm) compared with controls (302.0 ± 4.8 μm; P &lt; 0.0001), but did not undergo significant thinning until end-stage retinal degeneration (43.1 ± 6.5 μm). CONCLUSIONS. The data suggest that RPE loss is the primary cause of photoreceptor degeneration in choroideremia. The choroid is thinner than controls from early stages, in keeping with a mild developmental defect. Photoreceptors appear to lose outer segments following loss of underlying RPE and form tubulations at the edges of degeneration. The preservation of tubulations over time and after subretinal injection would be consistent with these structures maintaining attachment to the inner retina and hence being potentially light responsive (ClinicalTrials.gov, NCT01461213)

Real-world refractive outcomes of toric intraocular lens implantation in a United Kingdom National Health Service setting

Abstract Background With increasing availability of toric intraocular lenses (IOL) for cataract surgery, real-world refractive outcome data is needed to aid the counselling of patients regarding lens choice. We aim to assess the outcomes of toric intraocular lens use in the non-specialist environment of a typical United Kingdom NHS cataract service. Methods A retrospective cohort study conducted at the Oxford Eye Hospital, Oxford University Hospitals NHS Foundation Trust, UK. All patients who received a toric IOL implant over a 10 months period. Patients underwent pre-operative corneal marking, phacoemulsification and toric IOL implantation. Biometry was obtained using a Zeiss IOLMaster 500 and the toric IOLs were selected using the manufacturers’ online calculators. Post-operative refractions were obtained from optometrist’s manifest refraction or by autorefraction. The outcome measures were post-operative unaided visual acuity (UVA), spherical equivalent refraction, cylindrical correction and all complications. Results Thirty-two eyes of 24 patients aged 21–86 years (mean 66.4, SD 14.5) were included. UVA was superior to pre-operative best-corrected visual acuity (BCVA) in 81% of eyes, same in 16% and inferior in 3%, resulting in a median improvement of 0.20 LogMAR (IQR 0.10 to 0.30). 56%, 81%, 94% and 100% of eyes were within ±0.5, ±1.0, ±1.5 and ±2.0 D of predicted spherical equivalent, respectively. Three (9%) eyes required further surgery to rectify significant IOL rotation. Conclusions Reduced cylindrical correction and improved UVA could be expected in the majority of patients undergoing toric IOL implantation. Patients should be counselled about the risk of lens rotation

The Role of Inflammation in Age-Related Macular Degeneration—Therapeutic Landscapes in Geographic Atrophy

Age-related macular degeneration (AMD) is the leading cause of vision loss and visual impairment in people over 50 years of age. In the current therapeutic landscape, intravitreal anti-vascular endothelial growth factor (anti-VEGF) therapies have been central to the management of neovascular AMD (also known as wet AMD), whereas treatments for geographic atrophy have lagged behind. Several therapeutic approaches are being developed for geographic atrophy with the goal of either slowing down disease progression or reversing sight loss. Such strategies target the inflammatory pathways, complement cascade, visual cycle or neuroprotective mechanisms to slow down the degeneration. In addition, retinal implants have been tried for vision restoration and stem cell therapies for potentially a dual purpose of slowing down the degeneration and restoring visual function. In particular, therapies focusing on the complement pathway have shown promising results with the FDA approved pegcetacoplan, a complement C3 inhibitor, and avacincaptad pegol, a complement C5 inhibitor. In this review, we discuss the mechanisms of inflammation in AMD and outline the therapeutic landscapes of atrophy AMD. Improved understanding of the various pathway components and their interplay in this complex neuroinflammatory degeneration will guide the development of current and future therapeutic options, such as optogenetic therapy

Preliminary results in 18 patients undergoing retinal gene therapy for X-linked retinitis pigmentosa with codon-optimized AAV8-RPGR

Purpose: To investigate the safety and efficacy of adeno-associated viral vector (AAV8) encoding retinitis pigmentosa GTPase regulator (RPGR) for X-linked retinitis pigmentosa (XLRP).Methods: This study (ClinicalTrials. gov ID NCT03116113) is being conducted in 2 Parts: Part I is a Phase 1 dose-escalation study to identify the maximum tolerated dose (MTD); Part II is a Phase 2/3, assessor-masked, dose-expansion study. In Part I, subjects with genetically confirmed RPGR-associated XLRP were administered a sub-retinal dose of AAV8-RPGR. The vector used a rhodopsin kinase promoter to drive expression of a codon-optimized sequence that generated the correct full length RPGR protein. A 3+ 3 escalation scheme was used, with 3 subjects/dose (5× 10 9, 1× 10 10, 5× 10 10, 1× 10 11, 2.5× 10 11, and 5× 10 11 gp). Part I endpoints were safety (adverse events [AEs], dose-limiting toxicities [DLTs], ophthalmic