1,721,339 research outputs found
Pre−clinical evaluation of biocompatible nanoparticles as delivery system of 2O−methyl−phosphorothioate (2OMePS) antisense oligoribonucleotides for exon skipping−mediated dystrophin restoration (Telethon GGPO9093)
No full textDuchenne muscular dystrophy (DMD) is a severe hereditary neurodegenerative disorder due to mutations in the dystrophin gene. Effectiveness of antisense−mediated targeted exon
skipping for inducing dystrophin rescue has been demonstrated in a pilot trial in vivo by using a naked 2'OMePs antisense oligoribonucleotide. Other DMD PhaseI/II trials using backboned
PMO antisense are ongoing. We demonstrated as proof of principle that a novel biocompatible type of nanoparticle (T1,
with a core of polymethilmethacrylate) was able to bind and convey 2'OMePS antisense and to induce dystrophin restoration in body wide mdx animal model. In order to get insights onto
nanoparticles as AONs vehicles we will explore systemically in mdx mice: 1) other nanoparticles for identifying the best nanoparticle−AON compound as well as the optimal AON concentration able to have a sustained therapeutic effect; 2) different routes of administration; 3) clearance modes and possible side effects of nanoparticle−AON complexes. Studying other nanoparticles with higher AON loading capacity for their ability to induce dystrophin rescue in the mdx animal model will also allow us to optimise the dose regimen since we have evidences that NP have a depot effect, with long lasting release of
AONs. For the most effective nanoparticle we will perform pharmacokinetic studies of the nanoparticle−AON complex in order to assess the clearance mode and timing and the
half−life of these nanomaterials in the mdx animal model. We will also test different administration routes, including oral, since the known protective effect that nanoparticles exert on AON molecules. Furthermore we will evaluate the safety aspects of these novel compounds in regard to the sarcolemma components. This work will be performed in order to establish if nanoparticle−antisense complex might represent suitable, safe and efficacious compounds to be possibly transferred into DMD therapies
Modulazione di mutazioni del gene distrofina in vitro e in vivo mediante oligonucleotidi antisenso allo scopo di indurre exon skipping e ripristino della sintesi della proteina. (FAR 2007)
No full textIl progetto che si avvale di un’ampia serie di pazienti con distrofinopatia (piu di 150) si propone di: 1) valutare la capacità di legame fra diversi tipi di nanoparticelle e molecole antisenso a RNA 2) modulare il processo di splicing mediante nanoparticelle e RNA antisenso in cellule stabilizzate da tessuti di pazienti (cellule muscolari o della cute indotte a differenziare in senso miogenico) 3) modulare il processo di splicing mediante nanoparticelle e RNA antisenso in modelli animali (topo mdx e hamster). Tale approccio verrà utilizzato sia per indurre “exon skipping” in mutazioni frequenti come delezioni di vari esoni che per indurre lo “skipping” esonico di esoni contenenti mutazioni rare o private causa di fenotipo clinico severo, che possano essere indotte ad un comportamento di splicing più favorevole ed associato ad un fenotipo cellulare più lieve. Sottolineiamo come le nanoparticellle polimeriche da noi proposte abbiamo già mostrato assenza di tossicità e siano quindi appropriate ad un eventuale uso terapeutico per via sistemica anche nell’uomo. I risultati attesi riguardano la validazione della nostra ipotesi lavorativa, che mira ad ottenere complessi con AONs e nanoparticelle in gradi di raggiungere il cuore oltre che il muscolo scheletrico. Saremo inoltre in grado di valutare l’efficienza con cui le nostre molecole indurranno il fenomeno dello skipping esonico e il conseguente ripristino della proteina nelle cellule e animali trattati.
La modulazione di mutazioni con oligonucleotidi antisenso rappresenta un approccio molto promettente per possibili applicazioni terapeutiche. La valutazione della possibilità di modulare il maggior numero possibile di mutazioni distrofina e la ricerca di molecole in grado di migliorare il rilascio delle molecole antisenso rappresenta il prerequisito per impostare eventuali trial terapeutici
Ottimizzazione della strategia di diagnosi genetica per la distrofia muscolare di Duchenne (MIUR, Programma di collaborazione scientifica e tecnologica tra Italia e Albania per il periodo 2008-2010)
No full textLa DMD è una patologia ereditaria dovuta a mutazioni del gene distrofina e quindi legata geneticamente al cromosoma X. Come altre patologie mendeliane con ricaduta importante sulla definizione del genotipo e per la correlazione genotipo-fenotipo è caratterizzate da grande eterogeneità allelica. Questo rende complessa e multi-tecnologica la strategia diagnostica.Gli obiettivi del progetto sono:
Consulenza Genetica
-impostare un percorso di consulenza Genetica presso il Centro di Tirana in linea con le linee guida europee e tendo conto degli obiettivi di base che sono: i) valutare la componente genetica di una distrofinopatia; ii) identificare il modello ereditario; iii) valutare i rischi di ricorrenza/occorrenza; iv) indirizzare e allestire i test genetici specifici per la diagnosi genetica e la definizione del genotipo; v) impostare il monitoraggio riproduttivo; vi) supportare le decisioni rirpoduttive e informative autonome da parte dei pazienti e loro familiari; vii) interpretare i risultati dei test genetici alla luce dei progressi scientifici e delle possibilità di intervento preventivo.
Diagnosi Molecolare
- identificazione della mutazione causale nel gene distrofina eseguita nel laboratorio di Genetica Medica dell’Università di Ferrara. Tale identificazione rappresenta il prerequisito per allestire opportuni interventi preventivi, per la diagnosi delle femmine portatrici e per eventuali monitoraggi riproduttivi. Visto l’avanzamento scientifico e la presenza a breve di possibilità terapeutiche, la consulenza genetica e la diagnosi genetica della DMD rappresenta momento fondamentale nella gestione del paziente
Antisense modulation and characterisation of myogenic cells from 14 boys with Duchenne muscular dystrophy as pre-trial study aimed at patients’ recruitment for an European multicentric clinical trial (Telethon UILDM-GGPO7011)
No full textObjectives
Primary objectives of our project are i) to characterise myogenic cells from 14 DMD boys with dystrophin
deletions capable of being modulated by means of exon 46 and 51 skipping, in order to make them eligible
for the approaching European multicentric trial; ii) approval from the Istituto Superiore di Sanità of the
GMP molecules which will be used in the multicentric trials. Secondary objective is the evaluation of
dystrophin transcriptome changes in patients’ cells.
Background/Rationale
Antisense oligoribonucleotides are able to induce exon skipping in the dystrophin hnRNA therefore
by−passing out−of−frame deletions. The rationale of this approach is based on the fact that the majority of
DMDs is due to large deletions causing the disruption of the frame. The AONs exon skipping generates
internally deleted, but functional dystrophin proteins and would convert a severe DMD into a milder
phenotype. A successful first−in−man trial has recently been completed on DMD and a multicentric trial is
approaching.
Description of the project
We aim at performing on patient’s cells transfected with AONs the following studies: a) Real−Time
RT−PCR in order to finely detect and quantify non−skipped as well as skipped transcripts; b) functional
effect of the AONs treatment by evaluating dystrophin protein rescue; c) effect of AONs modulation on the
cell transcriptome by using an array−based strategy able to both define the dystrophin transcription atlas
and verify the correct dystrophin full mRNA composition after the treatment.
Genomic studies will be carried for mutation confirmation and SNPs occurrence.
Anticipated output
These studies will fully characterise AONs−treated cells of the selected Italian DMD patients making them
eligible and facilitating their inclusion in the first multicentric AONs trial for dystrophinopathies. The ISS
audit will allow us to obtain permission and ethical approval for the trial
'Nanoparticella del tipo core-shell idonea per la veicolazione di oligonucleotidi terapeutici in tessuti bersaglio e suo impiego per la preparazione di un medicamento per il trattamento della distrofia muscolare di Duchenne'
No full textLa presente invenzione riguarda una nanoparti-cella del tipo nucleo-guscio (core-shell) efficace nella veicolazione di oligonucleotidi terapeutici all’interno di cellule eucariotichetessuti bersaglio, e in particolarmente in cellule muscolari. L’invenzione riguarda inoltre un complesso nanoparticella/oligonucleotide terapeutico atto ad essere impiegato quale principio attivo in un medicamento, particolarmente un medicamento per il trattamento terapeutico della distrofia mu-scolare di Duchenne. L’invenzione può essere usata come medicamento in distrofinopatie Duchenne dovute a mutazioni per delezione esonica out-of-frame (con perdita della cornice di lettura) e/o piccole mutazioni in esoni in-frame (con cornice di lettura mantenuta) per ripristinare la cornice di lettura e quindi la sintesi di proteina distrofina
FUNCTIONAL ANALYISIS OF INTRONS AND GENE REGULATORY SEQUENCES AIMED AT TARGETED MODULATION OF GENE EXPRESSION (Progetto generale Eu FP5 STREP Finger, QLG2-CT-1999-00920)
No full textRegulation of gene expression is a complex phenomenon poorly understood. Our project aims to improve our knowledge
about non-coding sequences, increasingly shown to be relevant for modulating gene expression, using dystrophin gene as
a model.Among the series of characterised patients,available in the collaborative Units,several have unusual mutations and
phenotype suggesting a role of non-coding regions. We will analyse these patients in order to identify and functionally study
the regulatory sequences responsible.We will define the intronic breakpoints as well as other non-coding regions involved in
dystrophin mutations. We will characterise these sequences by experimental studies to test their regulatory function with
particular attention to those involved in pre-mRNA splicing. We will evaluate the feasibility of a targeted modulation of
dystrophin gene expression using antisense oligonucleotides
FP7-HEALTH-2012-INNOVATION-1 Neuromics
No full textNeurodegenerative (ND) and neuromuscular (NM) disease is one of the most frequent classes of rare diseases, affecting life and mobility of 500,000 patients in Europe and millions of their caregivers, family members and employers. This
NEUROMICS project brings together the leading research groups in Europe, five highly innovative SMEs and relevant oversea experts using the most sophisticated Omics technologies to revolutionize diagnostics and to develop pathomechanismbased
treatment for ten major ND and NM diseases. Specifically we aim to:
(i) use next generation WES to increase the number of known gene loci for the most heterogeneous disease groups from about 50% to 80%,
(ii) increase patient cohorts by large scale genotyping by enriched gene variant panels and NGS of so far unclassified patients and subsequent phenotyping,
(iii) develop biomarkers for clinical application with a strong emphasis on presymptomatic utility and cohort stratification,
(iv) combine -omics approaches to better understand pathophysiology and identify therapeutic targets,
(v) identify disease modifiers in disease subgroups cohorts with extreme age of onset
(vi) develop targeted therapies (to groups or personalized) using antisense oligos and histone deacetylase inhibitors, translating the consortiums expertise in clinical development from ongoing trials toward other disease groups, notably the PolyQ
diseases and other NMD.
To warrant that advances affect a large fraction of patients we limited the selection to a number of major categories, some of which are in a promising stage of etiological and therapeutic research while some others are in great need of further
classification. The efforts will be connected through a NEUROMICS platform for impact, communication and innovation that will provide tools and procedures for ensuring trial-readiness, WP performance, sustainability, interaction with the chosen
Definition of the dystrophin gene transcriptome and modulation of mutations by antisense oligonucleotides-induced targeted exon skipping (Telethon GGPO5115)
No full textAbstract
Dystrophin gene mutations cause dystrophinopathies, a group of phenotypes manifesting with striated muscle involvement. The expression of dystrophin is finely regulated and splicing plays a major role in modulating the phenotype. Recently both in vitro and in vivo modulation of dystrophin mutations by using antisense-mediated targeted exon skipping has been proven to be effective for restoring dystrophin synthesis, opening therapeutic perspectives. There are also increasing evidences that intronic regions contain regulatory elements as well as transcribe for non coding-RNA. The project, taking advantage of a large series of patients with dystrophinopathies available in our Unit, aims at: I) the identification and the characterisation of non–coding RNAs and related regulatory sequences possibly involved in dystrophinopathic phenotypes; ii) the modulation by antisense-mediated targeted exon skipping of dystrophin mutations in patient’s derived myogenic cells.
We will search for both conserved and non conserved transcripts within the dystrophin introns. We will investigate both their structural/functional characteristics and their relationship with dystrophin mutations/phenotypes by exploring their transcriptional status in patient’s tissues and by evaluating the occurrence in them of functional motifs. In selected patients with no mutations at the exonic level, we will explore the identified non-coding RNAs for the presence of causative mutations. The identification of as much as possible causative mutations/regulatory motifs in dystrophinopathies is a prerequisite in order to attempt a therapeutic approach for these devastating diseases. Antisense modulation in selected mutations in patient’s derived cells will allow us to restore the wild type RNA phenotype via targeted exon skipping. We will also test in patient’s cells a novel vehicle for antisense oligonucleotide delivery, the core-shell nanospheres, recently proposed as vaccine administration system
Diagnosi molecolare estensiva di distrofinopatia: una diagnosi per tutti (Progetto Generale Ente Duchenne Parent Project Italia)
No full textNostra intenzione è quella di identificare in modo estensivo tutte le mutazioni causali legate alle distrofinopatie.
A questo scopo verranno utilizzate diverse tecniche combinate quali MLPA , sequenziamento, analisi dell’ RNA, Real-time PCR e DHPLC che insieme consentono di identificare più del 95% delle mutazioni occorrenti nel gene distrofina.
Intendiamo inoltre allestire colture cellulare da muscolo di pazienti ( nei casi in cui sia proponibile a fini diagnostici una biopsia muscolare) al fine di impostare una modulazioni delle mutazioni identificate tramite antisenso. Intendiamo inoltre proporre in alcuni casi mirati biopsie cutanee atte ad ottenere fibroblasti che potranno essere convertiti in mioblasti tramite utilizzo di un fattore di trascrizione (MyoD) in grado di forzare il tipo cellulare. I mioblasti cosi ottenuti saranno quindi utilizzati sia a scopo diagnostico che al fine di modulare la mutazione in colture cellulari. Questo approccio rappresenta una importante alternativa alla biopsia muscolare ai fini della diagnostica su RNA. Sulle cellule disponibili (Muscolo o cute) valuteremo il coinvolgimento della proteina distrofina e delle proteine ad essa correlate con diagnosi immunoistochimica.
Abbiamo inoltre messo a punto un sistema diagnostico innovativo basato su array CGH per identificare delezioni duplicazione e riarrangiamenti nel gene distrofina localizzati anche in regioni non codificanti (introni, regioni regolative)
Identifying and validating pre-clinical biomarkers for diagnostics and therapeutics of Neuromuscular Disorders (BIO-NMD) (Grant agreement n.241665)
No full textThe rapidly expanding knowledge of NMDs genetic diagnosis, pathogenesis and therapeutic possibilities has provided new targets for disease characterisation, early diagnosis, drug discovery and development as well as has raised many questions about how to translate this knowledge into clinical practice as (initial) clinical trials typically run for such a short time that clinical improvement can hardly be expected within that time
frame. This militates for the discovery of surrogate endpoints for establishing the efficacy of clinical trials.
The concept of biomarkers represents measurable bio-parameters able to flank the process of diagnosis, functional characterisation and therapy in NMDs.
OMIC sciences (genomic, transcriptomics, proteomics) offer opportunities to identify biomarkers for finely defining and tuning the NMDs bases. This approach can make available non-invasive biomarkers, to be used for monitoring disease progression, prognosis and drugs response, therefore optimising the choice of
appropriate and often personalised therapies. The new genomic and proteomic biomarkers discovered within BIO-NMD will be validated both in animal models and in human samples, before entering into a qualification process at the EMEA. The qualified biomarkers resulting from the BIO-NMD project will be
ready for ongoing and further clinical trials for the patient benefit. This will increase the therapy efficacy and efficiency and also reduce adverse effects, with impact on patients’ quality of life with also economical relevance. The BIO-NMD consortium is led by UNIFE, which is an internationally recognised University in Genomic within hereditary neuromuscular disorders. In addition the consortium is composed of 7 leading European Academic partners bringing their expertise in all OMIC science as well as in bio-informatics and patient sample collection, 1 SME providing its skills in bio-informatics and 1 global company specialised in the
development of solution for patient samples screening
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