GP64-pseudotyped lentiviral vectors target liver endothelial cells and correct hemophilia A mice.

Lentiviral vectors (LV) are efficient vehicles for in vivo gene delivery to the liver. LV integration into the chromatin of target cells ensures their transmission upon proliferation, thus allowing potentially life-long gene therapy following a single administration, even to young individuals. The glycoprotein of the vesicular stomatitis virus (VSV.G) is widely used to pseudotype LV, as it confers broad tropism and high stability. The baculovirus-derived GP64 envelope protein has been proposed as an alternative for in vivo liver-directed gene therapy. Here, we perform a detailed comparison of VSV.G- and GP64-pseudotyped LV in vitro and in vivo. We report that VSV.G-LV transduced hepatocytes better than GP64-LV, however the latter showed improved transduction of liver sinusoidal endothelial cells (LSEC). Combining GP64-pseudotyping with the high surface content of the phagocytosis inhibitor CD47 further enhanced LSEC transduction. Coagulation factor VIII (FVIII), the gene mutated in hemophilia A, is naturally expressed by LSEC, thus we exploited GP64-LV to deliver a FVIII transgene under the control of the endogenous FVIII promoter and achieved therapeutic amounts of FVIII and correction of hemophilia A mice.

DEep LearnIng-based QuaNtification of epicardial adipose tissue predicts MACE in patients undergoing stress CMR.

BACKGROUND AND AIMS: This study investigated the additional prognostic value of epicardial adipose tissue (EAT) volume for major adverse cardiovascular events (MACE) in patients undergoing stress cardiac magnetic resonance (CMR) imaging. METHODS: 730 consecutive patients [mean age: 63 ± 10 years; 616 men] who underwent stress CMR for known or suspected coronary artery disease were randomly divided into derivation (n = 365) and validation (n = 365) cohorts. MACE was defined as non-fatal myocardial infarction and cardiac deaths. A deep learning algorithm was developed and trained to quantify EAT volume from CMR. EAT volume was adjusted for height (EAT volume index). A composite CMR-based risk score by Cox analysis of the risk of MACE was created. RESULTS: In the derivation cohort, 32 patients (8.7 %) developed MACE during a follow-up of 2103 days. Left ventricular ejection fraction (LVEF) < 35 % (HR 4.407 [95 % CI 1.903-10.202]; p<0.001), stress perfusion defect (HR 3.550 [95 % CI 1.765-7.138]; p<0.001), late gadolinium enhancement (LGE) (HR 4.428 [95%CI 1.822-10.759]; p = 0.001) and EAT volume index (HR 1.082 [95 % CI 1.045-1.120]; p<0.001) were independent predictors of MACE. In a multivariate Cox regression analysis, adding EAT volume index to a composite risk score including LVEF, stress perfusion defect and LGE provided additional value in MACE prediction, with a net reclassification improvement of 0.683 (95%CI, 0.336-1.03; p<0.001). The combined evaluation of risk score and EAT volume index showed a higher Harrel C statistic as compared to risk score (0.85 vs. 0.76; p<0.001) and EAT volume index alone (0.85 vs.0.74; p<0.001). These findings were confirmed in the validation cohort. CONCLUSIONS: In patients with clinically indicated stress CMR, fully automated EAT volume measured by deep learning can provide additional prognostic information on top of standard clinical and imaging parameters.

Predictors of adverse cardiac events of coronary myocardial bridging diagnosed with computed tomography angiography.

AIMS: Myocardial bridging (MB) is a frequent congenital anomaly of the epicardial coronary arteries commonly considered a benign condition. However, in some cases a complex interplay between anatomical, clinical and physiology factors may lead to adverse events, including sudden cardiac death. Coronary CT angiography (CCTA) emerged as the gold standard noninvasive imaging technique for the evaluation of MB. Aim of the study was to evaluate MB prevalence and anatomical features in a large population of patients who underwent CCTA for suspected CAD and to identify potential anatomical and clinical predictors of adverse cardiac events at long-term follow-up. METHODS AND RESULTS: Two-hundred and six patients (mean age 60.3 ± 11.8 years, 128 male) with MB diagnosed at CCTA were considered. A long MB was defined as ≥25 mm of overlying myocardium, whereas a deep MB as ≥2 mm of overlying myocardium. The study endpoint was the sum of the following adverse events: cardiac death, bridge-related acute coronary syndrome, hospitalization for angina or bridge-related ventricular arrhythmias and MB surgical treatment. Of the 206 patients enrolled in the study, 9 were lost to follow-up, whereas 197 (95.6%) had complete follow-up (mean 7.01 ± 3.0 years) and formed the analytic population. Nineteen bridge-related events occurred in 18 patients (acute coronary syndrome in 7, MB surgical treatment in 2 and hospitalization for bridge-related events in 10). Typical angina at the time of diagnosis and long MB resulted as significant independent predictors of adverse outcome. CONCLUSIONS: Typical angina and MB length ≥ 25 mm were independent predictors of cardiac events.

Quantifying Carotid Stenosis: History, Current Applications, Limitations, and Potential: How Imaging Is Changing the Scenario.

Carotid artery stenosis is a major cause of morbidity and mortality. The journey to understanding carotid disease has developed over time and radiology has a pivotal role in diagnosis, risk stratification and therapeutic management. This paper reviews the history of diagnostic imaging in carotid disease, its evolution towards its current applications in the clinical and research fields, and the potential of new technologies to aid clinicians in identifying the disease and tailoring medical and surgical treatment.

Partial correction of immunodeficiency by lentiviral vector gene therapy in mouse models carrying Rag1 hypomorphic mutations.

Introduction: Recombination activating genes (RAG) 1 and 2 defects are the most frequent form of severe combined immunodeficiency (SCID). Patients with residual RAG activity have a spectrum of clinical manifestations ranging from Omenn syndrome to delayed-onset combined immunodeficiency, often associated with granulomas and/or autoimmunity (CID-G/AI). Lentiviral vector (LV) gene therapy (GT) has been proposed as an alternative treatment to the standard hematopoietic stem cell transplant and a clinical trial for RAG1 SCID patients recently started. However, GT in patients with hypomorphic RAG mutations poses additional risks, because of the residual endogenous RAG1 expression and the general state of immune dysregulation and associated inflammation. Methods: In this study, we assessed the efficacy of GT in 2 hypomorphic Rag1 murine models (Rag1F971L/F971L and Rag1R972Q/R972Q), exploiting the same LV used in the clinical trial encoding RAG1 under control of the MND promoter. Results and discussion: Starting 6 weeks after transplant, GT-treated mice showed a decrease in proportion of myeloid cells and a concomitant increase of B, T and total white blood cells. However, counts remained lower than in mice transplanted with WT Lin- cells. At euthanasia, we observed a general redistribution of immune subsets in tissues, with the appearance of mature recirculating B cells in the bone marrow. In the thymus, we demonstrated correction of the block at double negative stage, with a modest improvement in the cortical/medullary ratio. Analysis of antigenspecific IgM and IgG serum levels after in vivo challenge showed an amelioration of antibody responses, suggesting that the partial immune correction could confer a clinical benefit. Notably, no overt signs of autoimmunity were detected, with B-cell activating factor decreasing to normal levels and autoantibodies remaining stable after GT. On the other hand, thymic enlargement was frequently observed, although not due to vector integration and insertional mutagenesis. In conclusion, our work shows that GT could partially alleviate the combined immunodeficiency of hypomorphic RAG1 patients and that extensive efficacy and safety studies with alternative models are required before commencing RAG gene therapy in thesehighly complex patients.

Circulating Small Extracellular Vesicles Reflect the Severity of Myocardial Damage in STEMI Patients.

Circulating small extracellular vesicles (sEVs) contribute to inflammation, coagulation and vascular injury, and have great potential as diagnostic markers of disease. The ability of sEVs to reflect myocardial damage assessed by Cardiac Magnetic Resonance (CMR) in ST-segment elevation myocardial infarction (STEMI) is unknown. To fill this gap, plasma sEVs were isolated from 42 STEMI patients treated by primary percutaneous coronary intervention (pPCI) and evaluated by CMR between days 3 and 6. Nanoparticle tracking analysis showed that sEVs were greater in patients with anterior STEMI (p = 0.0001), with the culprit lesion located in LAD (p = 0.045), and in those who underwent late revascularization (p = 0.038). A smaller sEV size was observed in patients with a low myocardial salvage index (MSI, p = 0.014). Patients with microvascular obstruction (MVO) had smaller sEVs (p < 0.002) and lower expression of the platelet marker CD41-CD61 (p = 0.039). sEV size and CD41-CD61 expression were independent predictors of MVO/MSI (OR [95% CI]: 0.93 [0.87-0.98] and 0.04 [0-0.61], respectively). In conclusion, we provide evidence that the CD41-CD61 expression in sEVs reflects the CMR-assessed ischemic damage after STEMI. This finding paves the way for the development of a new strategy for the timely identification of high-risk patients and their treatment optimization.

Pre-TAVI aortic annulus sizing: comparison between manual and semi-automated new generation software measurements in operators with different experience.

OBJECTIVES: Aim of the study is to compare manual and semi-automatic measurements for aortic annulus assessment among different operators. METHODS: Eighty patients who underwent TAVI were retrospectively enrolled. The measurements manually performed by an experienced reader for aortic annulus (minimum and maximum diameters, perimeter, area), annulus-to-coronary ostia distance and time needed for the whole evaluation, were collected. The same operator (observer 1) and two less experienced readers (observer 2 and 3, with >5 years and 1 year of experience, respectively) assessed the same measurements using a semi-automatic software. Differences between manual and semi-automatic measurements, reading time and suggested valves size derived by CT were compared. RESULTS: Very good correlations were found between manual and software-aided measurements for aortic annulus area and perimeter in comparison with standard measurements for the three readers (ICC range 0.81-0.98). Good correlations were found for the distance with coronary ostia(0.75-0.79). The same area-derived prosthesis size for manual and semi-automatic measurements was selected in 96% of cases for observer 1; very good correlations were also found for observer 2 and 3 (ICC = 0.89 and 0.88, respectively). Using semi-automatic measurements, the mean time needed for CT images was significantly lower for observers 1 and 2 (1.50 and 1.72versus 3.14 min), respectively. CONCLUSIONS: Pre-TAVI CT using semi-automatic software allows accurate and reproducible measurements, reducing reconstruction time up to 50% and is reliable even for operators with different experience. ADVANCES IN KNOWLEDGE: The use of semi-automatic dedicated software for CT in TAVI planning is reliable even for operators without long time experience and allows accurate and reproducible measurements improving pre-TAVI workflow.

Lipid nanoparticles allow efficient and harmless ex vivo gene editing of human hematopoietic cells.

Ex vivo gene editing in T cells and hematopoietic stem/progenitor cells (HSPCs) holds promise for treating diseases. Gene editing encompasses the delivery of a programmable editor RNA or ribonucleoprotein, often achieved ex vivo via electroporation, and when aiming for homology-driven correction of a DNA template, often provided by viral vectors together with a nuclease editor. Although HSPCs activate a robust p53-dependent DNA damage response upon nuclease-based editing, the responses triggered in T cells remain poorly characterized. Here, we performed comprehensive multiomics analyses and found that electroporation is the main culprit of cytotoxicity in T cells, causing death and cell cycle delay, perturbing metabolism, and inducing an inflammatory response. Nuclease RNA delivery using lipid nanoparticles (LNPs) nearly abolished cell death and ameliorated cell growth, improving tolerance to the procedure and yielding a higher number of edited cells compared with using electroporation. Transient transcriptomic changes upon LNP treatment were mostly caused by cellular loading with exogenous cholesterol, whose potentially detrimental impact could be overcome by limiting exposure. Notably, LNP-based HSPC editing dampened p53 pathway induction and supported higher clonogenic activity and similar or higher reconstitution by long-term repopulating HSPCs compared with electroporation, reaching comparable editing efficiencies. Overall, LNPs may allow efficient and harmless ex vivo gene editing in hematopoietic cells for the treatment of human diseases.

Quantification of extracellular volume with cardiac computed tomography in patients with dilated cardiomyopathy.

BACKGROUND: Cardiac computed tomography (CCT) was recently validated to measure extracellular volume (ECV) in the setting of cardiac amyloidosis, showing good agreement with cardiovascular magnetic resonance (CMR). However, no evidence is available with a whole-heart single source, single energy CT scanner in the clinical context of newly diagnosed left ventricular dysfunction. Therefore, the aim of this study was to test the diagnostic accuracy of ECVCCT in patients with a recent diagnosis of dilated cardiomyopathy, having ECVCMR as the reference technique. METHODS: 39 consecutive patients with newly diagnosed dilated cardiomyopathy (LVEF <50%) scheduled for clinically indicated CMR were prospectively enrolled. Myocardial segment evaluability assessment with each technique, agreement between ECVCMR and ECVCCT, regression analysis, Bland-Altman analysis and interclass correlation coefficient (ICC) were performed. RESULTS: Mean age of enrolled patients was 62 â€‹± â€‹11 years, and mean LVEF at CMR was 35.4 â€‹± â€‹10.7%. Overall radiation exposure for ECV estimation was 2.1 â€‹± â€‹1.1 â€‹mSv. Out of 624 myocardial segments available for analysis, 624 (100%) segments were assessable by CCT while 608 (97.4%) were evaluable at CMR. ECVCCT demonstrated slightly lower values compared to ECVCMR (all segments, 31.8 â€‹± â€‹6.5% vs 33.9 â€‹± â€‹8.0%, p â€‹< â€‹0.001). At regression analysis, strong correlations were described (all segments, r â€‹= â€‹0.819, 95% CI: 0.791 to 0.844). On Bland-Altman analysis, bias between ECVCMR and ECVCCT for global analysis was 2.1 (95% CI: -6.8 to 11.1). ICC analysis showed both high intra-observer and inter-observer agreement for ECVCCT calculation (0.986, 95%CI: 0.983 to 0.988 and 0.966, 95%CI: 0.960 to 0.971, respectively). CONCLUSIONS: ECV estimation with a whole-heart single source, single energy CT scanner is feasible and accurate. Integration of ECV measurement in a comprehensive CCT evaluation of patients with newly diagnosed dilated cardiomyopathy can be performed with a small increase in overall radiation exposure.

Cardiac computed tomography: from anatomy to function.

Ischaemic heart disease (IHD) is one of the world's leading causes of morbidity and mortality. Likewise, the diagnosis and risk stratification of patients with coronary artery disease (CAD) have always been based on the detection of the presence and extent of ischaemia by physical or pharmacological stress tests with or without the aid of imaging methods (e.g. exercise stress, test, stress echocardiography, single-photon emission computed tomography, or stress cardiac magnetic resonance). These methods show high performance to assess obstructive CAD, whilst they do not show accurate power to detect non-obstructive CAD. The introduction into clinical practice of coronary computed tomography angiography, the only non-invasive method capable of analyzing the coronary anatomy, allowed to add a crucial piece in the puzzle of the assessment of patients with suspected or chronic IHD. The current review evaluates the technical aspects and clinical experience of coronary computed tomography in the evaluation of atherosclerotic burden with a special focus about the new emerging application such as functional relevance of CAD with fractional flow reserve computed tomography (CT)-derived (FFRct), stress CT perfusion, and imaging inflammatory makers discussing the strength and weakness of each approach.

Tolerogenic IL-10-engineered dendritic cell-based therapy to restore antigen-specific tolerance in T cell mediated diseases.

Tolerogenic dendritic cells play a critical role in promoting antigen-specific tolerance via dampening of T cell responses, induction of pathogenic T cell exhaustion and antigen-specific regulatory T cells. Here we efficiently generate tolerogenic dendritic cells by genetic engineering of monocytes with lentiviral vectors co-encoding for immunodominant antigen-derived peptides and IL-10. These transduced dendritic cells (designated DCIL-10/Ag) secrete IL-10 and efficiently downregulate antigen-specific CD4+ and CD8+ T cell responses from healthy subjects and celiac disease patients in vitro. In addition, DCIL-10/Ag induce antigen-specific CD49b+LAG-3+ T cells, which display the T regulatory type 1 (Tr1) cell gene signature. Administration of DCIL-10/Ag resulted in the induction of antigen-specific Tr1 cells in chimeric transplanted mice and the prevention of type 1 diabetes in pre-clinical disease models. Subsequent transfer of these antigen-specific T cells completely prevented type 1 diabetes development. Collectively these data indicate that DCIL-10/Ag represent a platform to induce stable antigen-specific tolerance to control T-cell mediated diseases.

Intravenous Contrast Material for Cardiac Computed Tomography: Results From the Open-label Multicenter, Multivendor Italian Registry of Contrast Material Use in Cardiac Computed Tomography.

PURPOSE: The Italian Registry of Contrast Material use in Cardiac Computed Tomography (iRCM-CCT) is a multicenter, multivendor, observational study on the use of contrast media (CM) in patients undergoing cardiac computed tomography (CCT). The aim of iRCM-CCT is to assess image quality and safety profile of intravenous CM compounds. MATERIALS AND METHODS: iRCM-CCT enrolled 1842 consecutive patients undergoing CCT (≥50 per site) at 20 cluster sites with the indication of suspected coronary artery disease. Demographic characteristics, CCT, and CM protocols, clinical indications, safety markers, radiation dose reports, qualitative (ie, poor vascular enhancement) and quantitative (ie, HU attenuation values) image parameters were recorded. A centralized coordinating center collected and assessed all image parameters. RESULTS: The cohort included 891 men and 951 women (age: 63±14 y, body mass index: 26±4 kg/m2) studied with ≥64 detector rows computed tomography scanners and different iodinated intravenous CM protocols and compounds (iodixanol, iopamidol, iohexol, iobitridol, iopromide, and iomeprol). The following vascular attenuation was reported: 504±147 HU in the aorta, 451±146 HU in the right coronary artery, 474±146 HU in the left main, 451±146 HU in the left anterior descending artery, and 441±149 HU in the circumflex artery. In 4% of cases the image quality was not satisfactory due to poor enhancement. The following adverse reactions to CM were recorded: 6 (0.3%) extravasations and 17 (0.9%) reactions (11 mild, 4 moderate, 2 severe). CONCLUSIONS: In a multicenter registry on CM use during CCT the prevalence of CM-related adverse reactions was very low. The appropriate use of CM is a major determinant of image quality.

Comprehensive Evaluation of Left Ventricle Dysfunction by a New Computed Tomography Scanner: The E-PLURIBUS Study.

BACKGROUND: Although cardiac magnetic resonance (CMR) is considered the gold standard for myocardial fibrosis detection, cardiac computed tomography (CCT) is emerging as a promising alternative. OBJECTIVES: The purpose of this study was to assess feasibility and diagnostic accuracy of a comprehensive functional and anatomical evaluation with CCT as compared with CMR in patients with newly diagnosed left ventricular dysfunction (LVD). METHODS: A total of 128 consecutive patients with newly diagnosed LVD were screened. Based on the exclusion criteria, 28 cases were excluded. CCT was performed within 10 days from CMR. Biventricular volumes and ejection fraction, and presence and pattern of delayed enhancement (DE), were determined, along with evaluation of coronary arteries among patients undergoing invasive angiography in the 6 months after CCT. RESULTS: Six cases were excluded because of claustrophobia at CMR. Among the 94 patients who formed the study population, the concordance between CCT and CMR in suggesting the cause of the LVD was high (94.7%, 89/94 patients) in the overall population and was 100% for identifying ischemic cardiomyopathy. The CCT diagnostic rate for DE assessment was also high (96.7%, 1,544/1,598 territories) and similar to that of CMR (97.4%; P = 0.345, CCT vs CMR). Moreover, CCT showed high diagnostic accuracy in the detection of DE (94.8%, 95% CI: 93.6%-95.8%) in a territory-based analysis. Biventricular volumes and function parameters as measured by CCT and CMR were similar, without significant differences with the exception of a modest difference in RV volume. CCT was confirmed to be accurate for assessing arterial coronary circulation. The mean radiation exposure of the whole CCT was 7.78 ± 2.53 mSv (0.84 ± 0.24 mSv for DE). CONCLUSIONS: CCT performed with low-dose whole-heart coverage scanner and high-concentration contrast agent appears an effective noninvasive tool for a comprehensive assessment of patients with newly diagnosed LVD.

Approach to the Patient with Acute Aortic Syndromes in Light of the New Consensus Statement on Multimodality Imaging in Thoracic Aortic Diseases.

Acute aortic syndromes comprise a range of interrelated conditions including aortic dissection, intramural hematoma, penetrating atherosclerotic ulcer, and contained or not contained aortic aneurysm rupture. These syndromes are potentially life threatening; therefore, a rapid and accurate diagnosis is crucial. A new Clinical Consensus Statement on Aortic and Peripheral Vascular Disease has recently been published, and we will try to highlight the main innovations in the document.

Editing T cell repertoire by thymic epithelial cell-directed gene transfer abrogates risk of type 1 diabetes development.

Insulin is the primary autoantigen (Ag) targeted by T cells in type 1 diabetes (T1D). Although biomarkers precisely identifying subjects at high risk of T1D are available, successful prophylaxis is still an unmet need. Leaky central tolerance to insulin may be partially ascribed to the instability of the MHC-InsB9-23 complex, which lowers TCR avidity, thus resulting in defective negative selection of autoreactive clones and inadequate insulin-specific T regulatory cell (Treg) induction. We developed a lentiviral vector (LV)-based strategy to engineer thymic epithelial cells (TECs) to correct diabetogenic T cell repertoire. Intrathymic (it) LV injection established stable transgene expression in EpCAM+ TECs, by virtue of transduction of TEC precursors. it-LV-driven presentation of the immunodominant portion of ovalbumin allowed persistent and complete negative selection of responsive T cells in OT-II chimeric mice. We successfully applied this strategy to correct the diabetogenic repertoire of young non-obese diabetic mice, imposing the presentation by TECs of the stronger agonist InsulinB9-23R22E and partially depleting the existing T cell compartment. We further circumscribed LV-driven presentation of InsulinB9-23R22E by micro-RNA regulation to CD45- TECs without loss of efficacy in protection from diabetes, associated with expanded insulin-specific Tregs. Overall, our gene transfer-based prophylaxis fine-tuned the central tolerance processes of negative selection and Treg induction, correcting an autoimmune prone T cell repertoire.

Liver-directed lentiviral gene therapy corrects hemophilia A mice and achieves normal-range factor VIII activity in non-human primates.

Liver gene therapy with adeno-associated viral (AAV) vectors delivering clotting factor transgenes into hepatocytes has shown multiyear therapeutic benefit in adults with hemophilia. However, the mostly episomal nature of AAV vectors challenges their application to young pediatric patients. We developed lentiviral vectors, which integrate in the host cell genome, that achieve efficient liver gene transfer in mice, dogs and non-human primates, by intravenous delivery. Here we first compare engineered coagulation factor VIII transgenes and show that codon-usage optimization improved expression 10-20-fold in hemophilia A mice and that inclusion of an unstructured XTEN peptide, known to increase the half-life of the payload protein, provided an additional >10-fold increase in overall factor VIII output in mice and non-human primates. Stable nearly life-long normal and above-normal factor VIII activity was achieved in hemophilia A mouse models. Overall, we show long-term factor VIII activity and restoration of hemostasis, by lentiviral gene therapy to hemophilia A mice and normal-range factor VIII activity in non-human primate, paving the way for potential clinical application.