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

[This corrects the article DOI: 10.1016/j.omtm.2019.05.012.].

Characterizing the cellular immune response to subretinal AAV gene therapy in the murine retina.

Although adeno-associated viral (AAV) vector-mediated retinal gene therapies have demonstrated efficacy, the mechanisms underlying dose-dependent retinal inflammation remain poorly understood. Here, we present a quantitative analysis of cellular immune response to subretinal AAV gene therapy in mice using multicolor flow cytometry with a panel of key immune cell markers. A significant increase in CD45+ retinal leukocytes was detected from day 14 post-subretinal injection of an AAV8 vector (1 × 109 genome copies) encoding green fluorescent protein (GFP) driven by a ubiquitous promoter. These predominantly consisted of infiltrating peripheral leukocytes including macrophages, natural killer cells, CD4 and CD8 T cells, and natural killer T cells; no significant change in resident microglia population was detected. This cellular response was persistent at 28 days and suggestive of type 1 cell-mediated effector immunity. High levels (80%) of GFP fluorescence were found in the microglia, implicating their role in viral antigen presentation and peripheral leukocyte recruitment. When compared against AAV.GFP in paired eyes, an equivalent dose of an otherwise identical vector encoding the human therapeutic transgene Rab-escort protein 1 (REP1) elicited a significantly diminished cellular immune response (4.2-fold; p = 0.0221). However, the distribution of immune cell populations remained similar, indicating a common mechanism of AAV-induced immune activation.

Immunomodulatory Effects of Hydroxychloroquine and Chloroquine in Viral Infections and Their Potential Application in Retinal Gene Therapy.

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.

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.

DHX15 regulates CMTR1-dependent gene expression and cell proliferation.

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 methyl-transferase 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.

Use of FLIR ONE Smartphone Thermography in Burn Wound Assessment.

OBJECTIVE: Forward-looking infrared (FLIR) thermography technology uses a handheld camera that measures skin infrared emissivity, captures photographs, and can be analyzed through specialized software. Forward-looking infrared images can be used to analyze and correlate burn wound temperature with burn depth, burn progression, and the number of days needed for healing. FLIR ONE is a miniature, smartphone-compatible thermal imaging camera that has been used to assess inflammation in diabetic foot ulcers, as well as locating perforators in flap surgery. However, FLIR ONE's reliability in burn wound assessment has not been evaluated. This case series investigates the accuracy of FLIR ONE in comparison with the widely used indocyanine green (ICG) angiography in assessing burn wounds. METHODS: Five acute third-degree burn wounds were assessed using ICG angiography and FLIR ONE imaging (infrared thermography) to determine burn extent before surgical intervention. Patients were taken to the operating room within 48 hours of presentation; FLIR ONE images were captured approximately 35 to 45 cm above the wound surface. Margins of unsalvageable tissue as determined by ICG and FLIR ONE were marked and compared. RESULTS: The area of unsalvageable tissue as determined by FLIR ONE closely corresponded to the area determined by ICG. FLIR ONE overestimated unsalvageable tissue margins by approximately 1 to 2 cm. The area estimated by ICG consistently overlapped with more than 90% of the area estimated by FLIR ONE. CONCLUSIONS: There is a strong correlation between FLIR ONE and ICG when assessing salvageable tissue in third-degree burn wounds. FLIR ONE maximizes the convenience and cost-effectiveness of infrared thermography technology but may overestimate unsalvageable tissue area. FLIR ONE is promising as an adjunct to current imaging modalities such as ICG but requires further study for comparison.

Ultrahigh Frequency Ultrasound Imaging of the Hand: A New Diagnostic Tool for Hand Surgery.

BACKGROUND: Ultrasonography is a cost-effective, noninvasive, and expedient imaging modality with numerous clinical applications. Conventional ultrasound uses transducers with frequencies that range from 5 to 12 MHz. However, ultrahigh frequency ultrasound (UHFUS) is capable of producing frequencies up to 70 MHz, which can achieve tissue resolution up to 30 µm. The purpose of our study is to present the capabilities of a novel technology and to describe its possible clinical applications for hand surgery. METHODS: The Vevo 2100 (VisualSonics, Toronto, Canada) system was used to perform all ultrasound exams. Four unique linear array transducers were employed. All studies were performed by the authors, who have no formal training in ultrasound techniques, on 5 healthy resident volunteers and 1 clinical patient under institutional review board approval. RESULTS: A series of 10 static images per participant and dynamic, real-time videos were obtained at various locations within the hand and wrist. UHFUS is capable of quickly and reliably imaging larger structures such as foreign bodies, soft tissue masses, and the flexor tendons, and diagnosing an array of pathologies within these structures. In addition, UHFUS can identify much finer structures such as the intimal layer of the arteries in the hand and individual fascicles within the digital nerves to provide data about vessel quality and vascular and neural pathologies. CONCLUSIONS: UHFUS is a novel technology that shows multiple advantages over conventional ultrasound for imaging the fine superficial structures of the hand and wrist, and can be deployed by the surgeon at the point of care.