104 research outputs found
Antisense RNA Design, Delivery, and Analysis
No full textThis open access volume gathers a variety of models, delivery systems, and approaches that can be used to assess RNA technology for exploiting antisense as a therapeutic intervention. Beginning with a section on the design of antisense technology and their delivery, the book continues by covering model systems developed to evaluate efficacy, both in vivo and in vitro, as well as methods to evaluate preclinically the toxicity associated with these new potential drugs, and intellectual property considerations. Written for the highly successful Methods in Molecular Biology series, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls. Authoritative and practical, Antisense RNA Design, Delivery, and Analysis provides basic knowledge and a large collection of methods to facilitate the work of newcomers to this vibrant and expanding field. This book was conceived thanks to the network DARTER (Delivery of Antisense RNA Therapeutics). DARTER is funded by the EU Cooperation of Science and Technology (COST), which aims to enhance interaction and collaborations between researchers in Europe and other countries
Antisense RNA Design, Delivery, and Analysis
No full textThis open access volume gathers a variety of models, delivery systems, and approaches that can be used to assess RNA technology for exploiting antisense as a therapeutic intervention. Beginning with a section on the design of antisense technology and their delivery, the book continues by covering model systems developed to evaluate efficacy, both in vivo and in vitro, as well as methods to evaluate preclinically the toxicity associated with these new potential drugs, and intellectual property considerations. Written for the highly successful Methods in Molecular Biology series, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls. Authoritative and practical, Antisense RNA Design, Delivery, and Analysis provides basic knowledge and a large collection of methods to facilitate the work of newcomers to this vibrant and expanding field. This book was conceived thanks to the network DARTER (Delivery of Antisense RNA Therapeutics). DARTER is funded by the EU Cooperation of Science and Technology (COST), which aims to enhance interaction and collaborations between researchers in Europe and other countries
Antisense RNA Design, Delivery, and Analysis
No full textThis open access volume gathers a variety of models, delivery systems, and approaches that can be used to assess RNA technology for exploiting antisense as a therapeutic intervention. Beginning with a section on the design of antisense technology and their delivery, the book continues by covering model systems developed to evaluate efficacy, both in vivo and in vitro, as well as methods to evaluate preclinically the toxicity associated with these new potential drugs, and intellectual property considerations. Written for the highly successful Methods in Molecular Biology series, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls. Authoritative and practical, Antisense RNA Design, Delivery, and Analysis provides basic knowledge and a large collection of methods to facilitate the work of newcomers to this vibrant and expanding field. This book was conceived thanks to the network DARTER (Delivery of Antisense RNA Therapeutics). DARTER is funded by the EU Cooperation of Science and Technology (COST), which aims to enhance interaction and collaborations between researchers in Europe and other countries
Molecular Therapies for Inherited Retinal Diseases
No full textFollowing the implementation of next-generation sequencing technologies (e.g., exome and genome sequencing) in molecular diagnostics, the majority of genetic defects underlying inherited retinal disease (IRD) can readily be identified. In parallel, opportunities to counteract the molecular consequences of these defects are rapidly emerging, providing hope for personalized medicine. ‘Classical’ gene augmentation therapy has been under study for several genetic subtypes of IRD and can be considered a safe and sometimes effective therapeutic strategy. The recent market approval of the first retinal gene augmentation therapy product (LuxturnaTM, for individuals with bi-allelic RPE65 mutations) by the FDA has not only demonstrated the potential of this specific approach, but also opened avenues for the development of other strategies. However, every gene—or even every mutation—may need a tailor-made therapeutic approach, in order to obtain the most efficacious strategy with minimal risks associated. In addition to gene augmentation therapy, other subtypes of molecular therapy are currently being designed and/or implemented, including splice modulation, DNA or RNA editing, optogenetics and pharmacological modulation. In addition, the development of proper delivery vectors has gained strong attention, and should not be overlooked when designing and testing a novel therapeutic approach. In this Special Issue, we aim to describe the current state of the art of molecular therapeutics for IRD, and discuss existing and novel therapeutic strategies, from idea to implementation, and from bench to bedside
Use of Small Animal Models for Duchenne and Parameters to Assess Efficiency upon Antisense Treatment
No full textDuchenne muscular dystrophy (DMD) is a rare genetic disease affecting 1 in 5000 newborn boys. It is caused by mutations in the DMD gene with a consequent lack of dystrophin protein that leads to deterioration of myofibers and their replacement with fibro-adipogenic tissue. Using antisense oligonucleotides (AONs) to modify out-of-frame mutations in the DMD gene, named exon skipping, is currently considered among the most promising treatments for DMD patients. The development of this strategy is rapidly moving forward, and AONs designed to skip exons 51 and 53 have received accelerated approval in the USA. In preclinical setting, the mdx mouse model, carrying a point mutation in exon 23 of the murine Dmd gene that prevents production of dystrophin protein, has emerged as a valuable tool, and it is widely used to study in vivo therapeutic approaches for DMD. Here we describe the methodology for intravenous delivery of AONs targeting dystrophin through tail vein of mdx mice. Furthermore, the most relevant functional analyses to be performed in living mice, and the most informative histopathological and molecular assays to evaluate the effect of this treatment are detailed.</p
CERKL, generació d’un model knockout de retinosi pigmentària i estudis funcionals
No full text[cat] La Retinosi Pigmentària (RP), la causa de ceguesa més freqüent en adults (1:4000), està originada per mutacions en més de 50 gens. Un d’aquests és CERKL, gen identificat l’any 2004 pel grup, l’estudi funcional del qual constitueix el tema d’aquesta Tesi Doctoral. Fins el moment, s’han descrit vàries mutacions en CERKL causants d’RP o de distròfia de cons i bastons (CRD). A nivell proteic, CERKL presenta un 50% de similitud amb la proteïna quinasa de ceramides (CERK) i comparteix el domini diacilglicerol quinasa (DAGK). Endemés, CERKL presenta diferents dominis proteics coneguts (senyals de localització (NLS) i d’exportació (NES) nuclear, així com un domini d’homologia a plextrina (PH)). Tot i que s’han identificat mutacions que indubtablement causen degeneració retinal, encara resta per descriure la seva funció fisiològica, així com la seva contribució a la patologia.
L’objectiu principal d’aquest treball ha estat la caracterització funcional del gen i la proteïna CERKL a partir d’assaigs in vitro, combinant estratègies in silico, cel•lulars i moleculars, profunditzant en les anàlisis quantitatives i qualitatives dels principals esfingolípids. Finalment, també s’ha construït i analitzat un model animal murí knockout, Cerkl -/-.
Els principals resultats obtinguts en aquest treball s’enumeren a continuació:
1.- A nivell transcripcional, s’han caracteritzat de forma exhaustiva les isoformes produïdes per CERKL en retina humana i murina, revelant un nivell de complexitat inesperat, amb més de 20 isoformes, degudes principalment a empalmament alternatiu, així com a la presència de, com a mínim, quatre promotors diferents amb especificitat tissular.
2.- Estudis cel•lulars de sobreexpressió mostren que CERKL exerceix un efecte protector davant l’apoptosi causada per estrès oxidatiu, mentre que les formes mutants no retenen aquesta funció. Encara que els mecanismes de protecció es desconeixen, aquests resultats apunten a una funció neuroprotectora a curt termini de CERKL als fotoreceptors, els quals estan sotmesos de manera continuada a estrès lumínic i oxidatiu.
3.- Estudis exhaustius i detallats mitjançant tècniques de TLC, HPLC i UPLC per l’anàlisi de lípids han demostrat que CERKL no fosforila les ceramides ni cap dels esfingolípids identificats fins el moment, sota les condicions utilitzades. Tampoc s’ha pogut observar una funció quinasa de proteïnes. Els resultats obtinguts en aquest treball apunten a que CERKL ha d’exercir una altra funció a la cèl•lula, molt probablement lligada al transport de lípids o a la regulació d’altres processos cel•lulars.
4.- La localització subcel•lular ha determinat que CERKL localitza, principalment, al reticle endoplasmàtic i a l’aparell de Golgi i, de manera minoritària, al nucli, mostrant una localització extremadament dinàmica que recolza la contribució de CERKL en processos de transport i senyalització, així com una multiplicitat funcional.
5.- Un dels aspectes claus d’aquesta Tesi ha estat el disseny, generació i estudi fenotípic a nivell transcripcional, cel•lular, morfològic, electroretinogràfic i lipidòmic del model animal Cerkl-/-. El ratolí Cerkl-/- és viable i fèrtil, i els nostres estudis preliminars no mostren una degeneració de retina severa. El fenotip dels ratolins KO mostra una alteració significativa i consistent dels potencials oscil•latoris als electroretinogrames, els quals indiquen una afectació de les cèl•lules ganglionars i/o amacrines. Aquestes dades concorden amb les anàlisis immunohistoquímiques i d’hibridació in situ en retines de ratolí WT i KO, on la màxima expressió de CERKL es detecta en les cèl•lules ganglionars, i de manera més moderada, en la capa nuclear interna i fotoreceptors.
En resum, CERKL és un gen que mostra una gran complexitat transcripcional i funcional. Els pròxims objectius abordaran l’estudi en detall del model animal generat, així com d’altres models en vies de construcció.[eng] The main aim of this thesis was the transcriptional and functional characterisation of CERKL, one of the many genes described so far that are responsible for Retinitis Pigmentosa (RP), the main genetic cause of blindness in adults (prevalence 1:4.000). In particular, we aimed to: i) characterise the complex CERKL transcription in human and mouse retinas, ii) analyse the subcellular localisation of the CERKL encoded proteins, including the truncated isoform R257X, generated by the most frequent mutant allele; iii) characterise the substrate and possible enzymatic role of the CERKL protein; iv) describe the role of CERKL in the protection against oxidative stress; and last but not least, v) design, create and initially characterise a mouse Cerkl-/- model (knockout) at the transcriptional, histological, immunohistochemical and lipidomic levels, focussing on the retina.
Our results showed a high transcriptional complexity of the CERKL gene in the human and murine retina, with an extremely dynamic subcellular localisation, indicating the possibility of different functional roles in each compartment. The overexpression of CERKL protects cells from induced oxidative stress, suggesting that CERKL might play an important role in the initial response against oxidative injuries. CERKL harbours a conserved lipid kinase domain and the high homology with CERK has suggested a possible involvement in the sphingolipid metabolism. However, up to date, no kinase activity has been detected and the CERKL function remains unclear. In order to elucidate the CERKL physiological function, study its role in the retina, and whether it is (or not) related to sphingolipid metabolism, a knockout mouse was generated. The murine model resulted in a knockdown more than a knockout model, as alternative previously unreported promoters directed basal expression of Cerkl. Initial characterisation of the animal model phenotype showed a consistent and non-progressive alteration of the oscillatory potentials in the electroretinograms, an increase of apoptosis and GFAP protein (gliosis marker), as well as a decrease in Brn3a protein (ganglion cell marker), although no gross morphological changes were detectable. All together, these results point to clear signs of functional alteration at the ganglion cell layer. To determine the CERKL involvement in the sphingolipid metabolism, lipidomic quantification was performed and the analyses of the results are currently in progress
Preface of Special Issue "Molecular Therapies for Inherited Retinal Diseases"
No full textContains fulltext :
218308.pdf (Publisher’s version ) (Open Access)Inherited retinal diseases (IRDs) are a group of progressive disorders that lead to severe visual impairment or even complete blindness. IRDs display a vast heterogeneity, clinically as well as genetically, with over 250 genes identified in which mutations can cause one or more clinical subtypes of IRD. Long considered incurable diseases, intense research over the last two decades, combined with major technological advancements, have enabled the development of the first therapeutic approaches for these diseases. The approval of LuxturnaTM (voretigene neparvovec), a gene augmentation therapy vector for RPE65-associated IRD, by the US Food and Drug Administration and the European Medicines Agency, is considered a true milestone in the field, and has led to the development of similar, or different therapeutic strategies for many other subtypes of IRD. Despite these major achievements, there are still many aspects that can-and need to-be improved, including more insights into the relationship between genetic variation and cellular dysfunction, optimization of the vectors and sequences used, improving delivery methods, as well as understanding and modulating the (local) immune response. In addition, the extreme rarity of some genetic subtypes of IRDs poses an enormous challenge on the development of novel therapies, in terms of e.g., costs and regulatory affairs.[...]
Applications of antisense oligonucleotides for the treatment of inherited retinal diseases
No full textModeling Splicing Variants Amenable to Antisense Therapy by Use of CRISPR-Cas9-Based Gene Editing in HepG2 Cells
No full textThe field of splice modulating RNA therapy has gained new momentum with FDA approved antisense-based drugs for several rare diseases. In vitro splicing assays with minigenes or patient-derived cells are commonly employed for initial preclinical testing of antisense oligonucleotides aiming to modulate splicing. However, minigenes do not include the full genomic context of the exons under study and patients' samples are not always available, especially if the gene is expressed solely in certain tissues (e.g. liver or brain). This is the case for specific inherited metabolic diseases such as phenylketonuria (PKU) caused by mutations in the liver-expressed PAH gene.Herein we describe the generation of mutation-specific hepatic cellular models of PKU using CRISPR/Cas9 system, which is a versatile and easy-to-use gene editing tool. We describe in detail the selection of the appropriate cell line, guidelines for design of RNA guides and donor templates, transfection procedures and growth and selection of single-cell colonies with the desired variant , which should result in the accurate recapitulation of the splicing defect
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