Current Issue : July - September Volume : 2019 Issue Number : 3 Articles : 5 Articles
Gene therapy using adeno-associated viral (AAV) vectors currently represents the most\npromising approach for the treatment of many inherited retinal diseases (IRDs), given AAVâ??s\nability to efficiently deliver therapeutic genes to both photoreceptors and retinal pigment\nepithelium, and their excellent safety and efficacy profiles in humans. However, one of the main\nobstacles to widespread AAV application is their limited packaging capacity, which precludes their\nuse from the treatment of IRDs which are caused by mutations in genes whose coding sequence\nexceeds 5 kb. Therefore, in recent years, considerable effort has been made to identify strategies to\nincrease the transfer capacity of AAV vectors. This review will discuss these new developed\nstrategies, highlighting the advancements as well as the limitations that the field has still to\novercome to finally expand the applicability of AAV vectors to IRDs due to mutations in large\ngenes....
Fabry disease (FD) is a rare inherited disorder characterized by a wide range of systemic\nsymptoms; it is particularly associated with cardiovascular and renal problems. Enzyme\nreplacement therapy and pharmacological chaperone migalastat are the only approved and\neffective treatment strategies for FD patients. It is well documented that alpha-galactosidase A\n(GLA) enzyme activity deficiency causes globotriaosylceramide (Gb3) accumulation, which plays a\ncrucial role in the etiology of FD. However, the detailed mechanisms remain unclear, and the lack\nof a reliable and powerful disease model is an obstacle. In this study, we created such a model by\nusing CRISPR/Cas9-mediated editing of GLA gene to knockout its expression in human embryonic\nstem cells (hESCs). The cardiomyocytes differentiated from these hESCs (GLA-null CMs) were\ncharacterized by the accumulation of Gb3 and significant increases of cell surface area, the\nlandmarks of FD-associated cardiomyopathy. Furthermore, we used mass spectrometry to compare\nthe proteomes of GLA-null CMs and parental wild type CMs and found that the Rab GTPases\ninvolved in exocytotic vesicle release were significantly downregulated. This caused impairment of\nautophagic flux and protein turnover, resulting in an increase of reactive oxygen species and\napoptosis. To summarize, we established a FD model which can be used as a promising tool to study\nhuman hypertrophic cardiomyopathy in a physiologically and pathologically relevant manner and\nto develop new therapies by targeting Rab GTPases signaling-related exosomal vesicles\ntransportation....
Many rare diseases course with affectation of neurosensory organs. Among them, the\nneuroepithelial retina is very vulnerable due to constant light/oxidative stress, but it is also the most\naccessible and amenable to gene manipulation. Currently, gene addition therapies targeting retinal\ntissue (either photoreceptors or the retinal pigment epithelium), as a therapy for inherited retinal\ndystrophies, use adeno-associated virus (AAV)-based approaches. However, efficiency and safety of\ntherapeutic strategies are relevant issues that are not always resolved in virus-based gene delivery and\nalternative methodologies should be explored. Based on our experience, we are currently assessing the\nnovel physical properties at the nanoscale of inorganic gold nanoparticles for delivering genes to the\nretinal pigment epithelium (RPE) as a safe and efficient alternative approach. In this work, we present\nour preliminary results using DNA-wrapped gold nanoparticles (DNA-gold NPs) for successful in vitro\ngene delivery on human retinal pigment epithelium cell cultures, as a proof-of-principle to assess its\nfeasibility for retina in vivo gene delivery. Our results show faster expression of a reporter gene in\ncells transfected with DNA-gold NPs compared to DNA-liposome complexes. Furthermore, we\nshow that the DNA-gold NPs follow different uptake, internalization and intracellular vesicle\ntrafficking routes compared to pristine NPs....
Dilated cardiomyopathy (DCM) is a myocardial disease of dogs and humans characterized\nby progressive ventricular dilation and depressed contractility and it is a frequent cause of heart\nfailure. Conventional pharmacological therapy cannot reverse the progression of the disease and,\nin humans, cardiac transplantation remains the only option during the final stages of heart failure.\nCytoprotective gene therapy with vascular endothelial growth factor-B167 (VEGF-B167) has proved\nan effective alternative therapy, halting the progression of the disease in experimental studies on\ndogs. The aim of this work was to test the tolerability and feasibility of intracoronary administration,\nunder fluoroscopic guidance, of VEGF-B167 carried by adeno-associated viral vectors in canine DCM\npatients. Ten patients underwent the gene delivery procedure. The intraoperative phase was well\ntolerated by all dogs. Clinical and echocardiographic assessments at 7- and 30-days post-procedure\nshowed stable conditions compared to the pre-procedure phase. The results of this work indicate\nthat intracoronary VEGF-B167 gene delivery is feasible and tolerated in dogs with DCM. Further\nmonitoring/investigations are ongoing to evaluate the effects of this therapy on disease progression....
In addition to the protein coding information, viral RNA genomes code functional\ninformation in structurally conserved units termed functional RNA domains. These RNA domains\nplay essential roles in the viral cycle (e.g., replication and translation). Understanding the molecular\nmechanisms behind their function is essential to understanding the viral infective cycle. Further,\ninterfering with the function of the genomic RNA domains offers a potential means of developing\nantiviral strategies. Aptamers are good candidates for targeting structural RNA domains. Besides its\npotential as therapeutics, aptamers also provide an excellent tool for investigating the functionality\nof RNA domains in viral genomes. This review briefly summarizes the work carried out in our\nlaboratory aimed at the structural and functional characterization of the hepatitis C virus (HCV)\ngenomic RNA domains. It also describes the efforts we carried out for the development of antiviral\naptamers targeting specific genomic domains of the HCV and the human immunodeficiency virus\ntype-1 (HIV-1)....
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