Careful adjustment of Epo non-viral gene therapy for β-thalassemic anaemia treatment

BACKGROUND: In situ production of a secreted therapeutic protein is one of the major gene therapy applications. Nevertheless, the plasmatic secretion peak of transgenic protein may be deleterious in many gene therapy applications including Epo gene therapy. Epo gene transfer appears to be a promising alternative to recombinant Epo therapy for severe anaemia treatment despite polycythemia was reached in many previous studies. Therefore, an accurate level of transgene expression is required for Epo application safety. The aim of this study was to adapt posology and administration schedule of a chosen therapeutic gene to avoid this potentially toxic plasmatic peak and maintain treatment efficiency. The therapeutic potential of repeated muscular electrotransfer of light Epo-plasmid doses was evaluated for anaemia treatment in β-thalassemic mice. METHODS: Muscular electrotransfer of 1 μg, 1.5 μg, 2 μg 4 μg or 6 μg of Epo-plasmid was performed in β-thalassemic mice. Electrotransfer was repeated first after 3.5 or 5 weeks first as a initiating dose and then according to hematocrit evolution. RESULTS: Muscular electrotransfer of the 1.5 μg Epo-plasmid dose repeated first after 5 weeks and then every 3 months was sufficient to restore a subnormal hematrocrit in β-thalassemic mice for more than 9 months. CONCLUSION: This strategy led to efficient, long-lasting and non-toxic treatment of β-thalassemic mouse anaemia avoiding the deleterious initial hematocrit peak and maintaining a normal hematocrit with small fluctuation amplitude. This repeat delivery protocol of light doses of therapeutic gene could be applied to a wide variety of candidate genes as it leads to therapeutic effect reiterations and increases safety by allowing careful therapeutic adjustments

Defining A European Engineer Profile Within A European University Alliance

The world needs more engineers and Europe provides a rich and diverse environment to train them, including shared values of sustainability and interculturalism. In this paper we attempt to build a profile for a “European engineer” based on skills and competences acquired in a European University Alliance centred around engineering education (EELISA, European Engineering Learning Innovation Alliance). We carried out an on-line survey for students and staff of partner universities as well as nine indepth interviews (50 min) with relevante stakeholders. The questions included in the survey are described as well as general results from 75 respondents. The overall results from the in-depth interviews are also presented and discussed within the framework of the training concepts also promoted by international associations, including SEFI. Finally, we use our findings to suggest four conceptual fields for a European engineer profile: 1) Scientific and theoretical knowledge including digital skills, 2) Addressing sustainability, 3) Interculturalism: an engineer embracing the European project, and 4) Business and communication skills: practical and applied knowledge

In silico Design of an Epitope-Based Vaccine Ensemble for Chagas Disease

Trypanosoma cruzi infection causes Chagas disease, which affects 7 million people worldwide. Two drugs are available to treat it: benznidazole and nifurtimox. Although both are efficacious against the acute stage of the disease, this is usually asymptomatic and goes undiagnosed and untreated. Diagnosis is achieved at the chronic stage, when life-threatening heart and/or gut tissue disruptions occur in ∼30% of those chronically infected. By then, the drugs’ efficacy is reduced, but not their associated high toxicity. Given current deficiencies in diagnosis and treatment, a vaccine to prevent infection and/or the development of symptoms would be a breakthrough in the management of the disease. Current vaccine candidates are mostly based on the delivery of single antigens or a few different antigens. Nevertheless, due to the high biological complexity of the parasite, targeting as many antigens as possible would be desirable. In this regard, an epitope-based vaccine design could be a well-suited approach. With this aim, we have gone through publicly available databases to identify T. cruzi epitopes from several antigens. By means of a computer-aided strategy, we have prioritized a set of epitopes based on sequence conservation criteria, projected population coverage of Latin American population, and biological features of their antigens of origin. Fruit of this analysis, we provide a selection of CD8+ T cell, CD4+ T cell, and B cell epitopes that have <70% identity to human or human microbiome protein sequences and represent the basis toward the development of an epitope-based vaccine against T. cruzi

Placental Growth Factor Contributes to Micro-Vascular Abnormalization and Blood-Retinal Barrier Breakdown in Diabetic Retinopathy

OBJECTIVE: There are controversies regarding the pro-angiogenic activity of placental growth factor (PGF) in diabetic retinopathy (DR). For a better understanding of its role on the retina, we have evaluated the effect of a sustained PGF over-expression in rat ocular media, using ciliary muscle electrotransfer (ET) of a plasmid encoding rat PGF-1 (pVAX2-rPGF-1). MATERIALS AND METHODS: pVAX2-rPGF-1 ET in the ciliary muscle (200 V/cm) was achieved in non diabetic and diabetic rat eyes. Control eyes received saline or naked plasmid ET. Clinical follow up was carried out over three months using slit lamp examination and fluorescein angiography. After the control of rPGF-1 expression, PGF-induced effects on retinal vasculature and on the blood-external barrier were evaluated respectively by lectin and occludin staining on flat-mounts. Ocular structures were visualized through histological analysis. RESULTS: After fifteen days of rPGF-1 over-expression in normal eyes, tortuous and dilated capillaries were observed. At one month, microaneurysms and moderate vascular sprouts were detected in mid retinal periphery in vivo and on retinal flat-mounts. At later stages, retinal pigmented epithelial cells demonstrated morphological abnormalities and junction ruptures. In diabetic retinas, PGF expression rose between 2 and 5 months, and, one month after ET, rPGF-1 over-expression induced glial activation and proliferation. CONCLUSION: This is the first demonstration that sustained intraocular PGF production induces vascular and retinal changes similar to those observed in the early stages of diabetic retinopathy. PGF and its receptor Flt-1 may therefore be looked upon as a potential regulatory target at this stage of the disease

Applications of Plasmid Electrotransfer

The use of electric pulses to transfect cells has recently been extended to show the utility of this procedure in vivo. Electrotransfer has been performed in vivo on several tissue types including skin, blood vessels, liver, tumor, muscle, cornea, brain and spleen. The most widely targeted tissue has been skeletal muscle. In addition to its potential use in gene therapy, in vivo DNA electrotransfer is also, because of its simplicity, a powerful laboratory tool to study in vivo gene expression and function in a given tissue. Many published studies have now shown that plasmid electrotransfer can lead to a long-lasting therapeutic effect in various pathologies, such as cancer, blood disease, or muscle ischemia. The future potential for this gene therapy approach will include delivery for both local action or distal effect by secretion of the transgenic proteins in the circulation. </jats:p