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Transformative results of adeno-associated virus (AAV) gene therapy in patients with spinal muscular atrophy and Leber’s congenital amaurosis led to approval of the first two AAV products in the United States to treat these diseases. These extraordinary results led to a dramatic increase in the number and type of AAV gene-therapy programs. However, the field lacks non-invasive means to assess levels and duration of therapeutic protein function in patients. Here, we describe a new magnetic resonance imaging (MRI) technology for real-time reporting of gene-therapy products in the living animal in the form of an MRI probe that is activated in the presence of therapeutic protein expression. For the first time, we show reliable tracking of enzyme expression after a now in-human clinical trial AAV gene therapy (ClinicalTrials.gov: NTC03952637) encoding lysosomal acid beta-galactosidase (βgal) using a self-immolative βgal-responsive MRI probe. MRI enhancement in AAV-treated enzyme-deficient mice (GLB-1−/−) correlates with βgal activity in central nervous system and peripheral organs after intracranial or intravenous AAV gene therapy, respectively. With >1,800 gene therapies in phase I/II clinical trials (ClinicalTrials.gov), development of a non-invasive method to track gene expression over time in patients is crucial to the future of the gene-therapy field., Graphical abstract, We describe a bio-responsive contrast agent that informs on the biodistribution and durability of gene expression in the lysosomal storage disease, GM1 gangliosidosis. We illustrate the sensitivity of this contrast agent to detect β-galactosidase enzyme in the central nervous system and periphery of an AAV-GLB-1-treated mouse model of GM1.

GM1 gangliosidosis (GM1) is a fatal neurodegenerative lysosomal storage disease that occurs most commonly in young children, with no effective treatment available. Long-term follow-up of GM1 cats treated by bilateral thalamic and deep cerebellar nuclei (DCN) injection of adeno-associated virus (AAV)-mediated gene therapy has increased lifespan to 8 years of age, compared with an untreated lifespan of ~8 months. Due to risks associated with cerebellar injection in humans, the lateral ventricle was tested as a replacement route to deliver an AAVrh8 vector expressing feline β-galactosidase (β-gal), the defective enzyme in GM1. Treatment via the thalamus and lateral ventricle corrected storage, myelination, astrogliosis, and neuronal morphology in areas where β-gal was effectively delivered. Oligodendrocyte number increased, but only in areas where myelination was corrected. Reduced AAV and β-gal distribution were noted in the cerebellum with subsequent increases in storage, demyelination, astrogliosis, and neuronal degeneration. These postmortem findings were correlated with endpoint MRI and magnetic resonance spectroscopy (MRS). Compared with the moderate dose with which most cats were treated, a higher AAV dose produced superior survival, currently 6.5 years. Thus, MRI and MRS can predict therapeutic efficacy of AAV gene therapy and non-invasively monitor cellular events within the GM1 brain.

Osteoporosis is characterized by a loss of bone mass along with alterations in its structure, with subsequent increase in bone fragility and susceptibility to fractures, ultimately reinforcing the importance of its prevention by the reduction of the risk of falling.

Tay-Sachs Disease (TSD) is an inherited neurological disorder caused by deficiency of hexosaminidase A (HexA). Preclinical work demonstrated safety and efficacy of CNS gene therapy using AAVrh8-HEXA/HEXB. Here we describe an expanded access trial in two patients with infantile TSD (IND 18225). Case TSD-001 demonstrated neurodevelopmental regression by 8 months of age and severe seizures by 1 year was treated at 30 months. An equimolar mix of AAVrh8-HEXA and AAVrh8-HEXB (now AXO-AAV-GM2) was administered intrathecally (IT), with 75% of the dose (1x1014vg) delivered to the cisterna magna and 25% at the thoraco-lumbar junction. The second patient (TSD-002) was treated at 7 months of age with 4.2x1013 vg by a combination of bilateral thalamic (0.18 mL; 1.5x1012vg per thalamus), and IT infusion (3.9x1013vg). Both patients underwent immunosuppression with sirolimus, corticosteroids, and rituximab. Injection procedures were well tolerated and have shown no vector-related adverse events to date. CSF HexA activity nearly doubled from baseline and remained stable. In TSD-002 (now 16 months of age), MRI showed stabilization of disease by 3 months post-injection and appeared to temporarily deviate from the natural history of infantile TSD but declined again 6 months post-treatment. TSD-001 (now 4.5 years of age remains seizure-free on the same anti-convulsant therapy as pre-therapy, but TSD-002 developed seizures between 13 and 17 months posttreatment (by 2 years of age). Administration of AXO-AAV-GM2 by IT and thalamic injections was safe, HexA activity increased in CSF and ongoing myelination was apparent in the younger patient treated at an early symptomatic stage. This study provides early safety and proof-of-concept in humans for treatment of TSD patients by AAV gene therapy.

Goji berries’ bioactive compounds, which allowed classifying them as superfruits, led to an enormous increase of its consumption in western countries. However, the potential risk of allergy is a concern. In this study, we aimed to characterise the proteome of goji berries (Lycium barbarum) and identify proteins with putative role in the allergic reaction (IgE-binding proteins). We firstly used twodimensional (2D) gel electrophoresis followed by mass spectrometry (MS) to characterise goji berries’ proteome, and then Immunoblot reactivity with plasma from tomato and potato (same botanical family, Solanaceae) allergic individuals was assessed to characterise goji berries IgE-binding proteins. An inhibition assay was further performed to evaluate cross-reactivity among potato, tomato and goji berries. We significantly identified 93 out of the 180 MS analysed spots, corresponding to 29 protein functions. From these, 11 could be identified as goji berries IgE-binding proteins. We further demonstratedcross-reactivity between goji berries, tomato and potato. This work was supported by research unit GREEN-IT “Bioresources for Sustainability” (UID/Multi/04551/2013). IML acknowledges FCT for funding her PhD fellowship (PD/BD/113982/2015) under MolBioS PhD-Program (PD/00133/2012), and IAA acknowledges the FCT Investigator funding (POPH-QREN - IF/00764/2014). info:eu-repo/semantics/publishedVersion

GM1 gangliosidosis is a fatal neurodegenerative disease caused by a deficiency of lysosomal β-galactosidase. In its most severe form, GM1 gangliosidosis causes death by 4 years of age, and no effective treatments exist. Previous work has shown that injection of the brain parenchyma with an adeno-associated viral (AAV) vector provides pronounced therapeutic benefit in a feline GM1 model. To develop a less invasive treatment for the brain and increase systemic biodistribution, intravenous injection of AAV9 was evaluated. AAV9 expressing feline β-galactosidase was intravenously administered at 1.5×1013 vector genomes/kg body weight to six GM1 cats at ∼1 month of age. The animals were divided into two cohorts: (i) a long-term group, which was followed to humane end point; and (ii) a short-term group, which was analysed 16 weeks post-treatment. Clinical assessments included neurological exams, CSF and urine biomarkers, and 7 T MRI and magentic resonance spectroscopy (MRS). Post-mortem analysis included β-galactosidase and virus distribution, histological analysis and ganglioside content. Untreated GM1 animals survived 8.0 ± 0.6 months while intravenous treatment increased survival to an average of 3.5 years (n = 2) with substantial improvements in quality of life and neurological function. Neurological abnormalities, which in untreated animals progress to the inability to stand and debilitating neurological disease by 8 months of age, were mild in all treated animals. CSF biomarkers were normalized, indicating decreased CNS cell damage in the treated animals. Urinary glycosaminoglycans decreased to normal levels in the long-term cohort. MRI and MRS showed partial preservation of the brain in treated animals, which was supported by post-mortem histological evaluation. β-Galactosidase activity was increased throughout the CNS, reaching carrier levels in much of the cerebrum and normal levels in the cerebellum, spinal cord and CSF. Ganglioside accumulation was significantly reduced by treatment. Peripheral tissues such as heart, skeletal muscle, and sciatic nerve also had normal β-galactosidase activity in treated GM1 cats. GM1 histopathology was largely corrected with treatment. There was no evidence of tumorigenesis or toxicity. Restoration of β-galactosidase activity in the CNS and peripheral organs by intravenous gene therapy led to profound increases in lifespan and quality of life in GM1 cats. These data support the promise of intravenous gene therapy as a safe, effective treatment for GM1 gangliosidosis.

The GM2 gangliosidoses, Tay-Sachs disease (TSD) and Sandhoff disease (SD), are fatal lysosomal storage disorders caused by mutations in the HEXA and HEXB genes, respectively. These mutations cause dysfunction of the lysosomal enzyme β-N-acetylhexosaminidase A (HexA) and accumulation of GM2 ganglioside (GM2) with ensuing neurodegeneration, and death by 5 years of age. Until recently, the most successful therapy was achieved by intracranial co-delivery of monocistronic adeno-associated viral (AAV) vectors encoding Hex alpha and beta-subunits in animal models of SD. The blood-brain barrier crossing properties of AAV9 enables systemic gene therapy; however, the requirement of co-delivery of two monocistronic AAV vectors to overexpress the heterodimeric HexA protein has prevented the use of this approach. To address this need, we developed multiple AAV constructs encoding simultaneously HEXA and HEXB using AAV9 and AAV-PHP.B and tested their therapeutic efficacy in 4- to 6-week-old SD mice after systemic administration. Survival and biochemical outcomes revealed superiority of the AAV vector design using a bidirectional CBA promoter with equivalent dose-dependent outcomes for both capsids. AAV-treated mice performed normally in tests of motor function, CNS GM2 ganglioside levels were significantly reduced, and survival increased by >4-fold with some animals surviving past 2 years of age.

Global gene delivery to the CNS has therapeutic importance for the treatment of neurological disorders that affect the entire CNS. Due to direct contact with the CNS, cerebrospinal fluid (CSF) is an attractive route for CNS gene delivery. A safe and effective route to achieve global gene distribution in the CNS is needed, and administration of genes through the cisterna magna (CM) via a suboccipital puncture results in broad distribution in the brain and spinal cord. However, translation of this technique to clinical practice is challenging due to the risk of serious and potentially fatal complications in patients. Herein, we report development of a gene therapy delivery method to the CM through adaptation of an intravascular microcatheter, which can be safely navigated intrathecally under fluoroscopic guidance. We examined the safety, reproducibility, and distribution/transduction of this method in sheep using a self-complementary adeno-associated virus 9 (scAAV9)-GFP vector. This technique was used to treat two Tay-Sachs disease patients (30 months old and 7 months old) with AAV gene therapy. No adverse effects were observed during infusion or post-treatment. This delivery technique is a safe and minimally invasive alternative to direct infusion into the CM, achieving broad distribution of AAV gene transfer to the CNS.

Background Pharmacy-based interventions are complex public health endeavors which include, but are not restricted to, the conventional medication supply role. In diabetes, such interventions may improve patients’ outcomes. The aim of this study was to review relevant policies and research developed in Portugal directed at pharmacy-based diabetes interventions, and to inform future policies, practice and research in collaborative practice with primary care. Research method An exploratory review of diabetes legislation and policy papers, as well as a comprehensive review in Embase, MEDLINE (via Ovid and PubMed), Google Scholar, and grey literature until November 2017 was performed. Results Sixteen policy papers and 10 studies were included in the analysis. Positive evidence from pharmacy interventions was retrieved concerning screening individuals at risk, screening uncontrolled patients, managing diabetes, and supporting self-monitoring. Conclusions Some consistency in favorable findings, but also room for improvements in health policies, intervention design and research methods, were observed. Electronic supplementary material The online version of this article (10.1186/s40545-019-0166-1) contains supplementary material, which is available to authorized users.

O artigo foi elaborado a partir do estudo dos resultados de duas dissertações de mestrado (Estudo 1 e 2) e de uma tese de doutorado (Estudo 3). Foram identificadas reações emocionais comuns e freqüentes nos portadores de diabetes mellitus (DM), que comprometem o desempenho dos papéis de autocuidado. Teve como objetivos: apresentar as reações emocionais identificadas em portadores de DM tipo 1 e 2 e discutir as interferências das reações emocionais para desempenho dos papéis de autocuidado. Dos resultados, emergiram as categorias de reações emocionais: medo, raiva/ revolta, tristeza, depressão e estresse/ansiedade. Evidenciou-se ser a DM uma doença que, independente do tipo e da faixa etária, causa um impacto negativo biopsicossocial. Concluiu-se que há necessidade de uma abordagem mais ampla para a relação entre as reações emocionais e o desempenho das ações de autocuidado dos portadores de DM. The article was elaborated based on the study of the two thesis master of degrees (Study 1 and 2) and of a doctorate thesis (Study 3). Had been identified reactions emotional common and frequency in the patients with Diabetes Mellitus (DM), being able to compromise the action performance of papers of self-care. We have as objective: to present reactions emotional identified in patients with DM they intervene with the action performance of papers of self-care and arguing the emotional the interferences of for the self-care. Of the results the emotional categories of reactions had emerged: fear; anger, bad behavior, depressive feelings and stress/ anxiety. Evidencing to be independent DM an illness of the type and the age band it is the cause of a biopsychosocial negative impact. It is concluded the necessity of a larger approaching related to the emotional reaction and the action performance of self-care roles of patients with DM. El artículo fue elaborado basó en el estudio del año de dos tesis de bachillerato (estudio 1 y 2) y de una tesis del doctorado (el estudio 3). Habían sido el campo común y la frecuencia emocionales identificados de las reacciones en los pacientes con la Diabetes Mellitus (DM), pudiendo comprometer el funcionamiento de la acción de papeles del auto-cuidado. Tenemos como objetivo: para presentar a reacciones emocional identificado en pacientes con DM que él interviene con el funcionamiento de la acción de papeles del autocuidado y de discutir las interferencias para el uno mismo-cuidado. De los resultados las categorías emocionales de reacciones habían emergido: miedo; cólera, mal comportamiento, depresiva stress/ ansiedad . El evidenciar a ser la independiente DM a la enfermedad del tipo y la edad causa un impacto negativo biopsicosocial. Se concluí la necesidad de un amplio estudio para la relación entre las reacciones emocionales y el funcionamiento de la acción de papeles del auto-cuidado en pacientes con el DM.

Transthyretin (TTR) is a plasma and cerebrospinal fluid (CSF)-circulating homotetrameric protein. More than 100 point mutations have been identified in the TTR gene and several are related with amyloid diseases. Here we focused our attention in the TTR L12P variant associated with severe peripheral neuropathy and leptomeningeal amyloidosis. By using different cell lines derived from tissues specialized on TTR synthesis, such as the hepatocyte and the choroid plexus expressing WT, V30M, or L12P TTR variants we analyzed secretion, intracellular aggregation and degradation patterns. Also, we used liver-specific AAV gene transfer to assess expression of the L12P variant in vivo. We found the following: (i) decreased secretion with intracellular aggregation of TTR L12P in hepatoma cells relative to WT and V30M variant; this differential property of TTR L12P variant was also observed in mice injected with L12P AAV vector; (ii) differential N-glycosylation pattern of L12P variant in hepatoma cell lysates, conditioned media and mouse sera, which might represent an escape mechanism from ERAD degradation; (iii) intracellular L12P TTR aggregates mainly localized to lysosomes in cultured cells and liver; and (iv) none of the above findings were present in choroid plexus derived cells, suggesting particular secretion/quality control mechanisms that might contribute to leptomeningeal amyloidosis associated with the L12P variant. These observations open new avenues for the treatment of TTR associated leptomeningeal amyloidosis.

Carvedilol is a β-antagonist with strong anti-oxidant effect in lipids, proteins and superoxide production protecting biological membranes from oxidative stress. We hypothesised a possible therapeutical application of carvedilol in familial amyloidotic polyneuropathy (FAP), a neurodegenerative disease caused by deposition of transthyretin (TTR) amyloid fibrils. Oxidative stress, apoptosis and inflammation related to aggregation of non-fibrillar and fibrillar TTR have been detected both in pre-symptomatic and symptomatic stages of the disease, respectively. In this study we show by semi-quantitative immunohistochemistry and western blot analysis that administration of carvedilol to a transgenic mouse model of FAP influences the expression of oxidative and apoptotic biomarkers usually associated with TTR deposition, namely oxidation products such as HNE and 8-OHdG, and markers of ER stress. Most important, carvedilol treatment significantly reduced TTR aggregates in as much as 50%. We also observed that carvedilol rescues a mice neuroblastoma cell line from death induced by TTR oligomers, by decreasing activation of BiP and other ER stress biomarkers. Since carvedilol has no effect on TTR aggregation "in vitro", this finding points for the "in vivo" modulation of TTR aggregation by oxidative stress and apoptosis and prompts for the use of this safe drug in prophylactic and therapeutical measures in the FAP population.

The identification of specific biomarkers provides opportunities to develop early diagnostic parameters, monitor disease progression, and test drug efficiency in clinical trials. We previously demonstrated that in familial amyloidotic polyneuropathy (FAP) related to the abnormal extracellular tissue deposition of mutant transthyretin (TTR), inflammatory and apoptotic pathways are triggered in the presymptomatic stages of the disease, when nonfibrillar TTR deposits are present. In the present work, to better define biomarkers for future assessment of prophylactic and therapeutic drugs in the treatment of FAP, we extended the search for oxidative stress and apoptotic biomarkers to clinical samples and animal models presenting nonfibrillar and fibrillar TTR. We found that lipid peroxidation measured by hydroxynonenal, oxidative DNA damage measured by 8-hydroxy-2′-deoxyguanosine, and cellular redox homeostasis measured by glutaredoxin 1 were consistently increased in biopsy specimens from FAP patients and in tissues from transgenic mouse models presenting nonfibrillar TTR deposition. Death-receptor Fas, caspase-8, and Bax were also found to be increased, indicative of the involvement of death receptors in the observed apoptosis process. Removal of TTR deposition by an immunization protocol resulted in significant decreases of the selected markers we describe, corroborating the relationship between TTR deposition, oxidative stress, and apoptosis. Taken together, our results provide a robust biomarker profile for initial experimental animal studies and clinical trials to assess the application of the selected markers in therapies aimed at removal and/or inhibition of TTR polymerization.

Background: In spite of being among the main foods responsible for allergic reactions worldwide, soybean (Glycine max)-derived products continue to be increasingly widespread in a variety of food products due to their well-documented health benefits. Soybean also continues to be one of the elected target crops for genetic modification. The aim of this study was to characterize the soya proteome and, specifically, IgE-reactive proteins as well as to compare the IgE response in soya-allergic individuals to genetically modified Roundup Ready soya® versus its non-transgenic control. Methods: We performed two-dimensional gel electrophoresis of protein extracts from a 5% genetically modified Roundup Ready flour sample and its non-transgenic control followed by Western blotting with plasma from 5 soya-sensitive individuals. We used peptide tandem mass spectrometry to identify soya proteins (55 protein matches), specifically IgE-binding ones, and to evaluate differences between transgenic and non-transgenic samples. Results: We identified 2 new potential soybean allergens – one is maturation associated and seems to be part of the late embryogenesis abundant proteins group and the other is a cysteine proteinase inhibitor. None of the individuals tested reacted differentially to the transgenic versus non-transgenic samples under study. Conclusion: Soybean endogenous allergen expression does not seem to be altered after genetic modification. Proteomics should be considered a powerful tool for functional characterization of plants and for food safety assessment.

Background: The safety issues regarding foods derived from genetically modified (GM) plants are central to their acceptance into the food supply. The potential allergenicity of proteins newly introduced in GM foods is a major safety concern. Objective: We sought to monitor, in potentially sensitive human populations, the allergenicity effects of 5 GM materials obtained from sources with no allergenic potential and already under commercialization in the European Union. Methods: We have performed skin prick tests with protein extracts prepared from transgenic maize (MON810, Bt11, T25, Bt176) and soya (Roundup Ready) samples and from nontransgenic control samples in 2 sensitive groups: children with food and inhalant allergy and individuals with asthmarhinitis. We have also tested IgE immunoblot reactivity of sera from patients with food allergy to soya (Roundup Ready) and maize (MON810, Bt11, Bt176) samples, as well as to the pure transgenic proteins (CryIA[b] and CP4 5-enolpyruvylshikimate- 3-phosphate synthase). Results: None of the individuals undergoing tests reacted differentially to the transgenic and nontransgenic samples under study. None of the volunteers tested presented detectable IgE antibodies against pure transgenic proteins. Conclusion: The transgenic products under testing seem to be safe in terms of allergenic potential. We propose postmarket testing as an important screening strategy for putative allergic sensitization to proteins introduced in transgenic plants. Supported by Fundação Calouste Gulbenkian, research project SDH.SP.I.01.11 and by Comissão de Fomento da Investigação em Cuidados de Saúde, research project no. 186/01

Background: Allergies are nearly always triggered by protein molecules and the majority of individuals with documented immunologic reactions to foods exhibit IgE hypersensitivity reactions. In this study we aimed to understand if natural differences, at proteomic level, between maize populations, may induce different IgE binding proteins profiles among maize-allergic individuals. We also intended to deepen our knowledge on maize IgE binding proteins. Results: In order to accomplish this goal we have used proteomic tools (SDS-PAGE and 2-D gel electrophoresis followed by western blot) and tested plasma IgE reactivity from four maize-allergic individuals against four different protein fractions (albumins, globulins, glutelins and prolamins) of three different maize cultivars. We have observed that maize cultivars have different proteomes that result in different IgE binding proteins profiles when tested against plasma from maize-allergic individuals. We could identify 19 different maize IgE binding proteins, 11 of which were unknown to date. Moreover, we found that most (89.5%) of the 19 identified potential maize allergens could be related to plant stress. Conclusions: These results lead us to conclude that, within each species, plant allergenic potential varies with genotype. Moreover, considering the stress-related IgE binding proteins identified, we hypothesise that the environment, particularly stress conditions, may alter IgE binding protein profiles of plant components.