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Mutations in rhodopsin, the light-sensitive protein of rod cells, are the most common cause of autosomal dominant retinitis pigmentosa (ADRP). Many rod opsin mutations, such as P23H, lead to misfolding of rod opsin with detrimental effects on photoreceptor function and viability. Misfolded P23H rod opsin and other mutations in the intradiscal domain are characterized by the formation of an incorrect disulphide bond between C185 and C187, as opposed to the correct and highly conserved C110-C187 disulphide bond. Therefore, we tested the hypothesis that incorrect disulphide bond formation might be a factor that affects the biogenesis of rod opsin by studying wild-type (WT) or P23H rod opsin in combination with amino acid substitutions that prevent the formation of incorrect disulphide bonds involving C185. These mutants had altered traffic dynamics, suggesting a requirement for regulation of disulphide bond formation/reduction during rod opsin biogenesis. Here, we show that the BiP co-chaperone and reductase protein ERdj5 (DNAJC10) regulates this process. ERdj5 overexpression promoted the degradation, improved the endoplasmic reticulum mobility and prevented the aggregation of P23H rod opsin. ERdj5 reduction by shRNA delayed rod opsin degradation and promoted aggregation. The reductase and co-chaperone activity of ERdj5 were both required for these effects on P23H rod opsin. Furthermore, mutations in these functional domains acted as dominant negatives that affected WT rod opsin biogenesis. Collectively, these data identify ERdj5 as a member of the proteostasis network that regulates rod opsin biogenesis and supports a role for disulphide bond formation/reduction in rod opsin biogenesis and disease.

Inherited mutations in the rod visual pigment, rhodopsin, cause the degenerative blinding condition, retinitis pigmentosa (RP). Over 150 different mutations in rhodopsin have been identified and, collectively, they are the most common cause of autosomal dominant RP (adRP). Mutations in rhodopsin are also associated with dominant congenital stationary night blindness (adCSNB) and, less frequently, recessive RP (arRP). Recessive RP is usually associated with loss of rhodopsin function, whereas the dominant conditions are a consequence of gain of function and/or dominant negative activity. The in-depth characterisation of many rhodopsin mutations has revealed that there are distinct consequences on the protein structure and function associated with different mutations. Here we categorise rhodopsin mutations into seven discrete classes; with defects ranging from misfolding and disruption of proteostasis, through mislocalisation and disrupted intracellular traffic to instability and altered function. Rhodopsin adRP offers a unique paradigm to understand how disturbances in photoreceptor homeostasis can lead to neuronal cell death. Furthermore, a wide range of therapies have been tested in rhodopsin RP, from gene therapy and gene editing to pharmacological interventions. The understanding of the disease mechanisms associated with rhodopsin RP and the development of targeted therapies offer the potential of treatment for this currently untreatable neurodegeneration.

Objective.Rheumatoid arthritis (RA) patients’ adherence to disease-modifying antirheumatic drugs (DMARD) is often suboptimal. We examined associations among medication beliefs, self-efficacy, and adherence to medications in RA.Methods.Data were from a longitudinal observational cohort of persons with RA. Subjects completed telephone interviews on self-reported adherence, self-efficacy, demographics, and the Beliefs about Medicines Questionnaire (BMQ), which assesses beliefs in necessity and beliefs about taking medication. Bivariate and multivariate logistic regression identified correlates of poor adherence to synthetic DMARD and prednisone as well as to biologic therapy, including medication concerns and necessity.Results.There were 362 patients who reported taking a synthetic DMARD and/or prednisone. Of these, 14% and 21% reported poor adherence to oral DMARD or prednisone, and biologics, respectively. There were 64% who reported concern about taking medicines, 81% about longterm effects, and 47% about becoming too dependent on medicines. In multivariate analyses, the BMQ necessity score was independently associated with better adherence to oral DMARD or prednisone (adjusted OR 0.61, 95% CI 0.41–0.91), while self-efficacy was associated with greater odds of poor adherence to oral medications (adjusted OR 1.23, 95% CI 1.01–1.59). Beliefs in medicines and self-efficacy were not associated with adherence to biologics.Conclusion.In a diverse cohort of patients with RA, stronger beliefs in the necessity of medication were associated with better adherence to oral DMARD or prednisone, while higher self-efficacy was associated with poor adherence. Providers can play important roles in eliciting patient beliefs about medications to improve adherence and ultimately health outcomes.

Summary Background Mutations that disrupt the open reading frame and prevent full translation of DMD , the gene that encodes dystrophin, underlie the fatal X-linked disease Duchenne muscular dystrophy. Oligonucleotides targeted to splicing elements (splice switching oligonucleotides) in DMD pre-mRNA can lead to exon skipping, restoration of the open reading frame, and the production of functional dystrophin in vitro and in vivo, which could benefit patients with this disorder. Methods We did a single-blind, placebo-controlled, dose-escalation study in patients with DMD recruited nationally, to assess the safety and biochemical efficacy of an intramuscular morpholino splice-switching oligonucleotide (AVI-4658) that skips exon 51 in dystrophin mRNA. Seven patients with Duchenne muscular dystrophy with deletions in the open reading frame of DMD that are responsive to exon 51 skipping were selected on the basis of the preservation of their extensor digitorum brevis (EDB) muscle seen on MRI and the response of cultured fibroblasts from a skin biopsy to AVI-4658. AVI-4658 was injected into the EDB muscle; the contralateral muscle received saline. Muscles were biopsied between 3 and 4 weeks after injection. The primary endpoint was the safety of AVI-4658 and the secondary endpoint was its biochemical efficacy. This trial is registered, number NCT00159250. Findings Two patients received 0·09 mg AVI-4658 in 900 μL (0·9%) saline and five patients received 0·9 mg AVI-4658 in 900 μL saline. No adverse events related to AVI-4658 administration were reported. Intramuscular injection of the higher-dose of AVI-4658 resulted in increased dystrophin expression in all treated EDB muscles, although the results of the immunostaining of EDB-treated muscle for dystrophin were not uniform. In the areas of the immunostained sections that were adjacent to the needle track through which AVI-4658 was given, 44–79% of myofibres had increased expression of dystrophin. In randomly chosen sections of treated EDB muscles, the mean intensity of dystrophin staining ranged from 22% to 32% of the mean intensity of dystrophin in healthy control muscles (mean 26·4%), and the mean intensity was 17% (range 11–21%) greater than the intensity in the contralateral saline-treated muscle (one-sample paired t test p=0·002). In the dystrophin-positive fibres, the intensity of dystrophin staining was up to 42% of that in healthy muscle. We showed expression of dystrophin at the expected molecular weight in the AVI-4658-treated muscle by immunoblot. Interpretation Intramuscular AVI-4658 was safe and induced the expression of dystrophin locally within treated muscles. This proof-of-concept study has led to an ongoing systemic clinical trial of AVI-4658 in patients with DMD. Funding UK Department of Health.

Although antisense oligonucleotides (AO) have been used most commonly to inhibit gene expression, they can also be used to modify splicing of the primary transcript by targeting the exon splicing enhancers or the 3'/5' splice sites. Recent studies have shown that the morpholino chemistry is particularly useful as such AOs are not readily degraded. The mdx mouse is dystrophin deficient due to a premature stop codon in exon 23. Using an AO that targets exon 23, we have examined the longevity of exon skipping by RT-PCR and dystrophin transcript can still be detected 14 weeks after a single intramuscular injection of morpholino AO and dystrophin is clearly present at least 4 months post treatment. This is substantially longer than the expression seen in similar studies using a 2-O-methyl phosphorothioate AO. We have used similar techniques to examine the dose/volume relationship in preparation for clinical trials of this approach in Duchenne muscular dystrophy (DMD). As the diaphragm of the mdx mouse is the muscle that most closely resembles the pathology in DMD, exhibiting substantial fibrosis and fibre loss, we have used the diaphragm to test the effect of increased fibrosis in reducing AO uptake by the muscle fibres by treating mice at different ages. Delivery of AO in mice younger than 2 weeks can largely prevent the development of dystrophic pathology and treatment at later ages reduces the membrane fragility as determined by uptake of Evans Blue dye. These pre-clinical studies demonstrate the effectiveness of the morpholino based AO in inducing long-lived expression of dystrophin and this approach is now being taken forward to a UK clinical trial in DMD by the MDEX Consortium.