CMTR1 contributes to mRNA cap formation by methylating the first transcribed nucleotide ribose at the O-2 position. mRNA cap O-2 methylation has roles in mRNA stabilisation and translation, and self-RNA tolerance in innate immunity. We report that CMTR1 is recruited to serine-5–phosphorylated RNA Pol II C-terminal domain, early in transcription. We isolated CMTR1 in a complex with DHX15, an RNA helicase functioning in splicing and ribosome biogenesis, and characterised it as a regulator of CMTR1. When DHX15 is bound, CMTR1 activity is repressed and the methyltransferase does not bind to RNA pol II. Conversely, CMTR1 activates DHX15 helicase activity, which is likely to impact several nuclear functions. In HCC1806 breast carcinoma cell line, the DHX15–CMTR1 interaction controls ribosome loading of a subset of mRNAs and regulates cell proliferation. The impact of the CMTR1–DHX15 interaction is complex and will depend on the relative expression of these enzymes and their interactors, and the cellular dependency on different RNA processing pathways.

Effective treatment of retinal diseases with adeno-associated virus (AAV)-mediated gene therapy is highly dependent on the proportion of successfully transduced cells. However, due to inflammatory reactions at high vector doses, adjunctive treatment may be necessary to enhance the therapeutic outcome. Hydroxychloroquine and chloroquine are anti-malarial drugs that have been successfully used in the treatment of autoimmune diseases. Evidence suggests that at high concentrations, hydroxychloroquine and chloroquine can impact viral infection and replication by increasing endosomal and lysosomal pH. This effect has led to investigations into the potential benefits of these drugs in the treatment of viral infections, including human immunodeficiency virus and severe acute respiratory syndrome coronavirus-2. However, at lower concentrations, hydroxychloroquine and chloroquine appear to exert immunomodulatory effects by inhibiting nucleic acid sensors, including toll-like receptor 9 and cyclic GMP-AMP synthase. This dose-dependent effect on their mechanism of action supports observations of increased viral infections associated with lower drug doses. In this review, we explore the immunomodulatory activity of hydroxychloroquine and chloroquine, their impact on viral infections, and their potential to improve the efficacy and safety of retinal gene therapy by reducing AAV-induced immune responses. The safety and practicalities of delivering hydroxychloroquine into the retina will also be discussed.

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.

Retinitis pigmentosa (RP) is the most common cause of inherited retinal degeneration worldwide. It is characterized by the sequential death of rod and cone photoreceptors, the cells responsible for night and daylight vision, respectively. Although the expression of most RP genes occurs only in rods, there is a secondary degeneration of cones. One possible mechanism of cone death is metabolic dysregulation. Photoreceptors are highly metabolically active, consuming large quantities of glucose and producing substantial amounts of lactate. The retinal pigment epithelium (RPE) mediates the transport of glucose from the blood to photoreceptors and, in turn, removes lactate, which can influence the rate of consumption of glucose by the RPE. One model for metabolic dysregulation in RP suggests that following the death of rods, lactate levels are substantially diminished causing the RPE to withhold glucose, resulting in nutrient deprivation for cones. Here, we present adeno-associated viral vector-mediated delivery of monocarboxylate transporter 2 (MCT2, Slc16a7 ) into the eye, with expression limited to RPE cells, with the aim of promoting lactate uptake from the blood and encouraging the passage of glucose to cones. We demonstrate prolonged survival and function of cones in rat and mouse RP models, revealing a possible gene-agnostic therapy for preserving vision in RP. We also present the use of fluorescence lifetime imaging-based biosensors for lactate and glucose within the eye. Using this technology, we show changes to lactate and glucose levels within MCT2-expressing RPE, suggesting that cone survival is impacted by changes in RPE metabolism.

Retinitis pigmentosa is an inherited retinal disease caused by thousands of mutations in approximately 100 different genes. The most widely used mouse model for retinitis pigmentosa has the retinal degeneration 1 ( rd1 ) mutation in the Pde6b gene, which elicits rapid retinal degeneration and vision loss. A major limitation of these models is that these rd1 strains are not congenic, which prevents the use of appropriate controls. Furthermore, many strains have mutations in other genes which introduces genetic variability and may confound results. To address this issue, we backcrossed the rd1 allele from FVB mice onto a C57BL/6J genetic background over many generations, producing a C57BL/6J. Pde6b ^rd1 strain that was confirmed to be congenic to C57BL/6J mice. We show that this strain recapitulates the electroretinogram and optomotor results expected for mouse strains containing the rd1 mutation. Examination of retinal structure in cross sections of eyes isolated from C57BL/6J. Pde6b ^rd1 mice show a degree of thinning of the outer nuclear layer expected for a rd1 mutation, resulting in nearly complete loss of the outer nuclear layer by postnatal day 35. We anticipate that this C57BL/6J. Pde6b ^rd1 strain could become an asset for the field of retinitis pigmentosa research.

Retinitis pigmentosa (RP) is the most common cause of inherited retinal degeneration worldwide. It is characterized by the sequential death of rod and cone photoreceptors, the cells responsible for night and daylight vision, respectively. Although mutations in RP are mostly rod-specific, there is a secondary degeneration of cones. One possible mechanism behind cone death is metabolic dysregulation. Photoreceptors are highly metabolically active, consuming large quantities of glucose and producing substantial amounts of lactate. The retinal pigment epithelium (RPE) mediates the transport of glucose from the blood to photoreceptors and, in turn, removes lactate, which it can use as its own source of fuel. The model for metabolic dysregulation in RP suggests that, following the death of rods, lactate levels are substantially diminished causing the RPE to withhold glucose, resulting in nutrient deprivation for cones. Here, we present adeno-associated viral vector-mediated delivery of monocarboxylate transporter 2 (MCT2) into RPE cells with the aim of promoting lactate uptake from the blood and encouraging the passage of glucose to cones. We demonstrate prolonged survival and function of cones in rat and mouse RP models, revealing a possible gene agnostic therapy for preserving vision in RP. We also present the use of fluorescence lifetime imaging-based biosensors for lactate and glucose within the eye. Using this technology, we show changes to lactate and glucose levels within MCT2-expressing RPE, suggesting cone survival is impacted by RPE metabolism.