Light-responsive microRNA miR-211 targets Ezrin to modulate lysosomal biogenesis and retinal cell clearance.

Vertebrate vision relies on the daily phagocytosis and lysosomal degradation of photoreceptor outer segments (POS) within the retinal pigment epithelium (RPE). However, how these events are controlled by light is largely unknown. Here, we show that the light-responsive miR-211 controls lysosomal biogenesis at the beginning of light-dark transitions in the RPE by targeting Ezrin, a cytoskeleton-associated protein essential for the regulation of calcium homeostasis. miR-211-mediated down-regulation of Ezrin leads to Ca2+ influx resulting in the activation of calcineurin, which in turn activates TFEB, the master regulator of lysosomal biogenesis. Light-mediated induction of lysosomal biogenesis and function is impaired in the RPE from miR-211-/- mice that show severely compromised vision. Pharmacological restoration of lysosomal biogenesis through Ezrin inhibition rescued the miR-211-/- phenotype, pointing to a new therapeutic target to counteract retinal degeneration associated with lysosomal dysfunction.

Triple Vectors Expand AAV Transfer Capacity in the Retina.

Retinal gene transfer with adeno-associated viral (AAV) vectors holds great promise for the treatment of inherited retinal degenerations (IRDs). One limit of AAV is its transfer capacity of about 5 kb, which can be expanded to about 9 kb, using dual AAV vectors. This strategy would still not suffice for treatment of IRDs such as Usher syndrome type 1D or Alström syndrome type I (ALMS) due to mutations in CDH23 or ALMS1, respectively. To overcome this limitation, we generated triple AAV vectors, with a maximal transfer capacity of about 14 kb. Transcriptomic analysis following triple AAV transduction showed the expected full-length products along a number of aberrant transcripts. However, only the full-length transcripts are efficiently translated in vivo. We additionally showed that approximately 4% of mouse photoreceptors are transduced by triple AAV vectors and showed correct localization of recombinant ALMS1. The low-photoreceptor transduction levels might justify the modest and transient improvement we observe in the retina of a mouse model of ALMS. However, the levels of transduction mediated by triple AAV vectors in pig retina reached 40% of those observed with single vectors, and this bodes well for further improving the efficiency of triple AAV vectors in the retina.

Fully Liquid MenACWY-CRM Vaccine: Results from an Integrated Safety Analysis.

INTRODUCTION: The currently licensed quadrivalent MenACWY-CRM conjugate vaccine presentation consists of two vials (lyophilized MenA and liquid MenCWY) to be reconstituted before injection. A new fully liquid, single-vial formulation has been developed to simplify administration and prevent reconstitution errors. We present pooled safety data from two randomized, controlled, observer-blind phase 2b clinical trials, in which the fully liquid presentation was compared with the licensed presentation. METHODS: This is a post hoc analysis of two studies, in which safety data from participants aged 10-40 years who received one dose of either liquid MenACWY-CRM (1337 participants; MenACWY liquid group) or licensed MenACWY-CRM (1332 participants; MenACWY licensed group) were pooled. Frequencies were calculated for solicited adverse events (AEs) during 7 days post-vaccination and unsolicited AEs, including medically attended AEs and serious AEs (SAEs), during the 6-month safety follow-up period. Analysis results are presented by vaccine group, overall and by age category (10-17 and 18-40 years). RESULTS: Overall, AEs solicited for collection during the first 7 days after vaccination were reported by similar percentages of participants (69.2%, MenACWY liquid; 68.2%, MenACWY licensed), and were generally mild/moderate in intensity. Solicited local AEs were reported by 46.0% of the MenACWY liquid group and 43.5% of the MenACWY licensed group and solicited systemic AEs by 55.2 and 54.1%, respectively. During the 6-month post-vaccination period, unsolicited AEs were reported by 32.2 and 31.2% of the MenACWY liquid group and MenACWY licensed group, respectively, and medically attended AEs by 18.6 and 17.3%, respectively. Overall, 14 participants in each group (1.0 and 1.1%, respectively) reported SAEs, none of which was considered vaccine-related by the investigator. The safety profiles of both MenACWY-CRM presentations were similar for each age group and overall. CONCLUSIONS: This pooled analysis shows the safety profile of fully liquid MenACWY-CRM is comparable with that of the currently licensed vaccine presentation. CLINICAL TRIAL REGISTRATION: ClinicalTrials.gov Identifiers: NCT03652610 (August 29, 2018), NCT03433482 (14 February 2018).

Immunological non-inferiority of a new fully liquid presentation of the MenACWY-CRM vaccine to the licensed vaccine: results from a randomized, controlled, observer-blind study in adolescents and young adults.

A fully liquid MenACWY-CRM vaccine presentation has been developed, modifying the meningococcal serogroup A (MenA) component from lyophilized to liquid. The safety and immunogenicity of the liquid presentation at the end of the intended shelf-life (aged for 24 or 30 months) were compared to the licensed lyophilized/liquid presentation. This multicenter, randomized (1:1), observer-blind, phase 2b study (NCT03433482) enrolled adolescents and young adults (age 10-40 years). In part 1, 844 participants received one dose of liquid presentation stored for approximately 24 months or licensed presentation. In part 2, 846 participants received one dose of liquid presentation stored for approximately 30 months or licensed presentation. After storage, the MenA free saccharide (FS) level was approximately 25% and O-acetylation was approximately 45%. The primary objective was to demonstrate non-inferiority of the liquid presentation to licensed presentation, as measured by human serum bactericidal assay (hSBA) geometric mean titers (GMTs) against MenA, 1-month post-vaccination. Immune responses against each vaccine serogroup were similar between groups. Between-group ratios of hSBA GMTs for MenA were 1.21 (part 1) and 1.11 (part 2), with two-sided 95% confidence interval lower limits (0.94 and 0.87, respectively) greater than the prespecified non-inferiority margin (0.5), thus meeting the primary study objective. No safety concerns were identified. Despite reduced O-acetylation of MenA and increased FS content, serogroup-specific immune responses induced by the fully liquid presentation were similar to those induced by the licensed MenACWY-CRM vaccine, with non-inferior anti-MenA responses. The safety profiles of the vaccine presentations were similar.

Intein-mediated protein trans-splicing expands adeno-associated virus transfer capacity in the retina.

Retinal gene therapy with adeno-associated viral (AAV) vectors holds promises for treating inherited and noninherited diseases of the eye. Although clinical data suggest that retinal gene therapy is safe and effective, delivery of large genes is hindered by the limited AAV cargo capacity. Protein trans-splicing mediated by split inteins is used by single-cell organisms to reconstitute proteins. Here, we show that delivery of multiple AAV vectors each encoding one of the fragments of target proteins flanked by short split inteins results in protein trans-splicing and full-length protein reconstitution in the retina of mice and pigs and in human retinal organoids. The reconstitution of large therapeutic proteins using this approach improved the phenotype of two mouse models of inherited retinal diseases. Our data support the use of split intein-mediated protein trans-splicing in combination with AAV subretinal delivery for gene therapy of inherited blindness due to mutations in large genes.

High-Throughput Screening Identifies Kinase Inhibitors That Increase Dual Adeno-Associated Viral Vector Transduction In Vitro and in Mouse Retina.

Retinal gene therapy based on adeno-associated viral (AAV) vectors is safe and efficient in humans. The low intrinsic DNA transfer capacity of AAV has been expanded by dual vectors where a large expression cassette is split in two halves independently packaged in two AAV vectors. Dual AAV transduction efficiency, however, is greatly reduced compared to that obtained with a single vector. As AAV intracellular trafficking and processing are negatively affected by phosphorylation, this study set to identify kinase inhibitors that can increase dual AAV vector transduction. By high-throughput screening of a kinase inhibitors library, three compounds were identified that increase AAV transduction in vitro, one of which has a higher effect on dual than on single AAV vectors. Importantly, the transduction enhancement is exerted on various AAV serotypes and is not transgene dependent. As kinase inhibitors are promiscuous, siRNA-mediated silencing of targeted kinases was performed, and AURKA and B, PLK1, and PTK2 were among those involved in the increase of AAV transduction levels. The study shows that kinase inhibitor administration reduces AAV serotype 2 (AAV2) capsid phosphorylation and increases the activity of DNA-repair pathways involved in AAV DNA processing. Importantly, the kinase inhibitor PF-00562271 improves dual AAV8 transduction in photoreceptors following sub-retinal delivery in mice. The study identifies kinase inhibitors that increase dual and single AAV transduction by modulating AAV entry and post-entry steps.