1,721,013 research outputs found
Transcriptional enhancement as therapeutic approach of coagulation factor VII promoter mutations
No full textThe main scope of the proposed project is to explore the ability of engineered Transcription Factors (eTFs) to rescue gene expression of F7 gene in the presence of mutations impairing gene transcription. More specific objectives are:
-to identify eTFs able to enhance F7 gene transcription in human liver cell lines,
-to identify eTFs able to rescue F7 gene transcription affected by mutations in cellular models,
-to generate mouse models of F7 promoter mutations
-to produce liver specific Adeno-Associated Virus (AAV) for the delivery of eTFs
-to provide the proof-of-principle for the eTFs mediated rescue of F7 gene expression in mouse models.
We expect to provide in vitro and in vivo insights into the ability of eTFs to rescue naturally occurring F7 promoter mutations and increase FVII expression levels. This method might suggest innovative therapeutic approaches for coagulation factor deficiencies or other human diseases caused by impaired gene transcription
Correction of duplications in the DMD gene by a CRISPR/Cas9 approach
No full textThe long term goal of this project is to generate the first therapeutic approach for tandem exon duplications. The selected
target will be the most frequent duplication in the dystrophin gene (DMD): exon 2 duplication. In order to open the way to a
therapy for this class of neglected variations we will develop viral vectors to test the efficacy of the approach in in vitro and in
vivo models. In detail, we have assembled CRISPR/Cas9 systems to target the DMD gene exon 2 duplication with the idea
to target intronic portions of the duplicated region creating two double strand breaks and stimulate the repair machinery to
recombine these sites leading to the exclusion of the duplication.
The aim of the project are: 1) testing of CRISPR/Cas9 activity in myogenic immortalized cells; 2) creation of a hDMD mdx
mouse model bearing exon 2 duplications; 3) AAV-CRISPR/Cas9 vector creation for the treatment of relevant DMD mouse
models; 4) assessment of the induced modifications and of rescue of dystrophin synthesis, 5) assessment of the off-target
cleavage by specific Cas9-ChIP protocol.
This proposal will evaluate the feasibility of a gene targeting approach to rescue the exon 2 duplication which results in a
DMD phenotype. Overall these objectives will provide the proof-of-principle for a therapeutic approach for duplications
paving the way for the treatment of several other diseases
Correction of duplications in the DMD gene by a CRISPR/Cas9 approach
No full textThe long term goal of this project is to generate the first therapeutic approach for tandem exon
duplications. The selected target will be the most frequent duplication in the dystrophin gene (DMD):
exon 2 duplication. In order to open the way to a therapy for this class of neglected variations we will
develop viral vectors to test the efficacy of the approach in in vitro models. In detail, we will: 1)
assemble CRISPR/Cas9 systems to target the DMD gene exon 2 duplication: the system will target an
intronic portion of the duplicated region so that it will create two double strand breaks and stimulate the
repair machinery to recombine these sites leading to the exclusion of the duplication; 2) deliver
CRISPR/Cas9 systems in HEK293 and myogenic cells to identify the more efficient one; 3) produce
immortalized myogenic cells from fibroblasts of patients with exon 2 duplications as cellular models
for testing our therapeutic approach; 4) develop Adeno-Associated Virus (AAV) vectors for the
delivery of the selected CRISPR/Cas9 systems in the myogenic cells derived from fibroblasts of
patients with exon 2 duplication and characterize the resulting effect.
This proposal will evaluate the feasibility of a gene targeting approach to rescue the exon 2 duplication
which results in a DMD phenotype. Overall these objectives will provide the proof-of-principle for a
therapeutic approach for duplications paving the way for the treatment of several other diseases
Targeted Genome Editing in Spinal Muscular Atrophy
No full textSpinal muscular atrophy (SMA) is an autosomal recessive neuromuscular disorder caused by mutations in the survival-motor-neuron 1 (SMN1) telomeric gene. Deficiencies in the ubiquitous SMN function affect multiple tissues and organs; however neuronal tissue is primarily sensitive, resulting in α-motor neuron degeneration in the ventral horn of the spinal cord with subsequent neuromuscular-junction dysfunction and proximal muscle weakness. The onset of disease and degree of severity are variable in patients and they are determined in part by multiple copies of the centromeric homologue SMN2 that inversely correlate with the phenotypic severity. Indeed, SMN2 gene mainly produces a truncated form SMN∆7 by aberrant alternative splicing and a small amount (~10%) of the fully active full-length SMN, thus buffering the SMN deficiency. A potential strategy for treating SMA patients is to increase SMN levels in the affected tissues, hence gene therapy and modifiers of SMN2-alternative splicing have proved therapeutic efficacy in SMA animal models.
In this study, we explored the possibility of applying targeted genome editing technology to the human SMN locus in order to revert the SMN2 sequence to a SMN1-like sequence that may undergo proper splicing under the the endogenous transcriptional control. The resulting correction would be permanent and lead to longlasting protein production in gene-edited cells. We used the streptococcus pyogenes Cas9-CRISPR system to target the SMN2 gene at different locations. Two main strategies were explored: i) SMN1_exon7 addition/correction by promoting homology-driven DNA repair, ii) SMN2_intron7_ intronic-splicing-silencer (ISS-N1) mutation and correction of SMN2 aberrant splicing, by exploiting the non-homologous end-joining (NHEJ) pathway. Plasmids encoding Cas9-GFP under the control of CMV promoter, and selected gRNAs downstream to the Pol-III U6 promoter (Addgene) were transfected in HEK-293T cell line and in immortalized myoblasts derived from either healthy donors or SMA patients. Transfection efficiency was estimated as percentage of GFP-expressing cells (20-50% and 1-10%, respectively) and nuclease activity detected by Surveyor assay and target site sequencing. In particular, in SMA patient-derived myoblasts we detected mutations (indels) at the level of the induced DNA double-strand break at ~30% frequency. Levels of SMN restoration will be investigated by qPCR of the different species of SMN transcripts and by western blotting of SMN protein. The goal of this study is to provide an in vitro proof of principle of effective gene correction in SMA patient-derived cells. In the context of a multisystemic, complicated disease such as SMA, targeted genome editing strategy could represent an additional therapeutic too
Rapid, comprehensive analysis of the dystrophin transcript by a custom micro-fluidic exome array
No full textDuchenne and Becker muscular dystrophies are caused by mutations in the dystrophin gene. Both the enormous size of this gene and heterogeneous set of causative mutations behind these pathologies may hamper and even prevent accurate molecular diagnosis. Often RNA analysis is required not only to identify mutations escaping MLPA/CGH or exon sequencing but also to validate the functional effect of novel variations that may affect the exon composition of the DMD gene. We present the design and experimental validation of a new, simple, and easy-to-use platform we call FluiDMD. This platform is based on the Applied Biosystems 7900HT TaqMan ® low-density array technology and is able to define the full-exon composition, profile the dystrophin isoforms present, establish changes in mRNA decay, and potentially identify all deletions/duplications and splicing affecting mutations contemporaneously. Moreover, we demonstrate that this system accurately detects the pathogenic effect of all dystrophin mutations belonging to any category, thereby highlighting the functional validation capacity of this system. The high efficacy and sensitivity of this tool in detecting mutations in the dystrophin transcript can be exploited in a variety of cells/tissues, in particular skin, which is harvested by causing minimum patient discomfort. We therefore propose FluiDMD as a validated diagnostic biomarker for molecular profiling of dystrophinopathies. © 2011 Wiley Periodicals, Inc
Crispr/Cas9-based COL7A1 editing for recessive dystrophic epidermolysis bullosa
No full textAbstract non disponibil
MOLECULAR MECHANISMS AND THERAPEUTIC APROACHES FOR RESTORATION OF mRNA TRANSCRIPTION, MATURATION AND TRANSLATION IN INHERITED COAGULATION FACTOR DEFICIENCIES
No full textAlthough substitutive therapy in coagulation factor deficiencies has recently evolved towards proteins with extended half-life and reduced risk for complications, the quality of life of patients would be significantly ameliorated by innovative approaches, based on alternative strategies and able to provide prolonged/permanent expression of therapeutic levels of the defective factor.
Among these, rescuing the altered pre-RNA processing/maturation and mRNA translation has received particular attention because it would in principle permit to restore expression of the mutated gene still maintaining its physiological regulation in the competent tissues, especially hepatocytes which efficiently deliver several coagulation proteins.
More recently, gene editing approaches permit specific DNA sequence recognition, cleavage and thus repair/correction of mutated genes in the appropriate cells.
Here we focus on molecular mechanisms supporting innovative approaches for restoring transcription, maturation and translation of mRNAs in inherited coagulation factor deficiencies:
By engineering transcription activator-like effectors fused with an activation domain (TALE-TFs), able to specifically rescue the coagulation factor VII promoter activity impaired by severe disease-causing mutations. In turn, this triggers synthesis of factor VII mRNA and secretion of functional factor VII protein.
By engineering the key component of the spliceosome, the small nuclear RNA U1 (U1 snRNA), able to prevent exon skipping in mutated factor VII and factor IX pre-mRNA exon-intron junctions. In turn, this triggers synthesis of correct mRNA and secretion of functional factors.
By aminoglycoside drugs inducing ribosome readthrough on premature translation termination codons affecting factor VII. This permits synthesis of full length protein with procoagulant function instead of truncated non-functional molecules.
Depending on the approach and mutations affecting patients' mRNA, we report in cellular and animal models expression levels ranging from negligible to the rescue of potentially therapeutic amounts of coagulation factor activity. Our data support further studies aimed at evaluating clinical translatability of specific molecules in selected groups of patients
Exploring Splicing-Switching Molecules For Seckel Syndrome Therapy
No full textThe c.2101 A > G synonymous change (p.G674G) in the gene for ATR, a key player in the DNA-damage response, has been the first identified genetic cause of Seckel Syndrome (SS), an orphan disease characterized by growth and mental retardation. This mutation mainly causes exon 9 skipping, through an ill-defined mechanism. Through ATR minigene expression studies, we demonstrated that the detrimental effect of this mutation (6 ± 1% of correct transcripts only) depends on the poor exon 9 definition (47 ± 4% in the ATRwt context), because the change was ineffective when the weak 5′ or the 3′ splice sites (ss) were strengthened (scores from 0.54 to 1) by mutagenesis. Interestingly, the exonic c.2101 A nucleotide is conserved across species, and the SS-causing mutation is predicted to concurrently strengthen a Splicing Silencer (ESS) and weaken a Splicing Enhancer (ESE). Consistently, the artificial c.2101 A > C change, predicted to weaken the ESE only, moderately impaired exon inclusion (28 ± 7% of correct transcripts). The observation that an antisense oligonucleotide (AONATR) targeting the c.2101 A position recovers exon inclusion in the mutated context supports a major role of the underlying ESS. A U1snRNA variant (U1ATR) designed to perfectly base-pair the weak 5'ss, rescued exon inclusion (63 ± 3%) in the ATRSS-allele. Most importantly, upon lentivirus-mediated delivery, the U1ATR partially rescued ATR mRNA splicing (from ~ 19% to ~ 54%) and protein (from negligible to ~ 6%) in embryonic fibroblasts derived from humanized ATRSS mice. Altogether these data elucidate the molecular mechanisms of the ATR c.2101 A > G mutation and identify two potential complementary RNA-based therapies for Seckel syndrome
An engineered tale-transcription factor rescues transcription of factor VII impaired by promoter mutations and enhances its endogenous expression in hepatocytes
No full textTailored approaches to restore defective transcription responsible for severe diseases have been poorly explored. We tested transcription activator-like effectors fused to an activation domain (TALE-TFs) in a coagulation factor VII (FVII) deficiency model. In this model, the deficiency is caused by the -94C > G or -61T > G mutation, which abrogate the binding of Sp1 or HNF-4 transcription factors. Reporter assays in hepatoma HepG2 cells naturally expressing FVII identified a single TALE-TF (TF4) that, by targeting the region between mutations, specifically trans-activated both the variant (>100-fold) and wild-type (20-40-fold) F7 promoters. Importantly, in the genomic context of transfected HepG2 and transduced primary hepatocytes, TF4 increased F7 mRNA and protein levels (2- to 3-fold) without detectable off-target effects, even for the homologous F10 gene. The ectopic F7 expression in renal HEK293 cells was modestly affected by TF4 or by TALE-TF combinations. These results provide experimental evidence for TALE-TFs as gene-specific tools useful to counteract disease-causing promoter mutations
Prenatal diagnosis of Duchenne muscular dystrophy by comparative genomic hybridization
No full textDuchenne muscular dystrophy (DMD, OMIM#310200) is a severe neuromuscular disorder caused by mutations in the dystrophin gene; it has a prevalence of 1 in 3500 live males (1–2).
Due to the enormous size of this gene, and to allele heterogeneity, molecular diagnosis of DMD requires a great deal of effort. While multiplex ligation-dependent probe amplification (MLPA) represents the standard molecular technique for detecting exonic DMD gene rearrangements (3), several comparative genomic hybridization (CGH) platforms have recently been reported to rapidly screen the entire DMD gene, as well as neighboring sequences, for deletions and duplications (4–6)
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