Biomedical subjects

Olivier Danos

Publications and source records attributed to Olivier Danos.

At least 19 recordsLinked to original sources

PubMed2007

Multiexon skipping leading to an artificial DMD protein lacking amino acids from exons 45 through 55 could rescue up to 63% of patients with Duchenne muscular dystrophy.

Approximately two-thirds of Duchenne muscular dystrophy (DMD) patients show intragenic deletions ranging from one to several exons of the DMD gene and leading to a premature stop codon. Other deletions that maintain the translational reading frame of the gene result in the milder Becker muscular dystrophy (BMD) form of the disease. Thus the opportunity to transform a DMD phenotype into a BMD phenotype appeared as a new treatment strategy with the development of antisense oligonucleotides technology, which is able to induce an exon skipping at the pre-mRNA level in order to restore an open reading frame. Because the DMD gene contains 79 exons, thousands of potential transcripts could be produced by exon skipping and should be investigated. The conventional approach considers skipping of a single exon. Here we report the comparison of single- and multiple-exon skipping strategies based on bioinformatic analysis. By using the Universal Mutation Database (UMD)-DMD, we predict that an optimal multiexon skipping leading to the del45-55 artificial dystrophin (c.6439_8217del) could transform the DMD phenotype into the asymptomatic or mild BMD phenotype. This multiple-exon skipping could theoretically rescue up to 63% of DMD patients with a deletion, while the optimal monoskipping of exon 51 would rescue only 16% of patients.

Adolescent↗

PubMed2007

Phenotypic correction of alpha-sarcoglycan deficiency by intra-arterial injection of a muscle-specific serotype 1 rAAV vector.

alpha-Sarcoglycanopathy (limb-girdle muscular dystrophy type 2D, LGMD2D) is a recessive muscular disorder caused by deficiency in alpha-sarcoglycan, a transmembrane protein part of the dystrophin-associated complex. To date, no treatment exists for this disease. We constructed recombinant pseudotype-1 adeno-associated virus (rAAV) vectors expressing the human alpha-sarcoglycan cDNA from a ubiquitous or a muscle-specific promoter. Evidence of specific immune response leading to disappearance of the vector was observed with the ubiquitous promoter. In contrast, efficient and sustained transgene expression with correct sarcolemmal localization and without evident toxicity was obtained with the muscle-specific promoter after intra-arterial injection into the limbs of an LGMD2D murine model. Transgene expression resulted in restoration of the sarcoglycan complex, histological improvement, membrane stabilization, and correction of pseudohypertrophy. More importantly, alpha-sarcoglycan transfer produced full rescue of the contractile force deficits and stretch sensibility and led to an increase of the global activity of the animals when both posterior limbs are injected. Our results establish the feasibility for AAV-mediated alpha-sarcoglycan gene transfer as a therapeutic approach.

Animals↗

PubMed2006

CUG-BP1/CELF1 requires UGU-rich sequences for high-affinity binding.

CUG-BP1 [CUG-binding protein 1 also called CELF (CUG-BP1 and ETR3 like factors) 1] is a human RNA-binding protein that has been implicated in the control of splicing and mRNA translation. The Xenopus homologue [EDEN-BP (embryo deadenylation element-binding protein)] is required for rapid deadenylation of certain maternal mRNAs just after fertilization. A variety of sequence elements have been described as target sites for these two proteins but their binding specificity is still controversial. Using a SELEX (systematic evolution of ligand by exponential enrichment) procedure and recombinant CUG-BP1 we selected two families of aptamers. Surface plasmon resonance and electrophoretic mobility-shift assays showed that these two families differed in their ability to bind CUG-BP1. Furthermore, the selected high-affinity aptamers form two complexes with CUG-BP1 in electrophoretic mobility assays whereas those that bind with low affinity only form one complex. The validity of the distinction between the two families of aptamers was confirmed by a functional in vivo deadenylation assay. Only those aptamers that bound CUG-BP1 with high affinity conferred deadenylation on a reporter mRNA. These high-affinity RNAs are characterized by a richness in UGU motifs. Using these binding site characteristics we identified the Xenopus maternal mRNA encoding the MAPK (mitogen-activated protein kinase) phosphatase (XCl100alpha) as a substrate for EDEN-BP. In conclusion, high-affinity CUG-BP1 binding sites are sequence elements at least 30 nucleotides in length that are enriched in combinations of U and G nucleotides and contain at least 4 UGU trinucleotide motifs. Such sequence elements are functionally competent to target an RNA for deadenylation in vivo.

3' Untranslated Regions↗

PubMed2006

Characterization of the gene transfer process mediated by histidine-rich peptides.

Peptides have an interesting potential as gene transfer agents not only because they are biodegradable but also due to their reduced size and the facility of product characterization and large-scale production. Recently, we have shown that the cationic amphipathic 26-mer peptide LAH4 possesses high DNA delivery capacities. To better understand its way of functioning and to further improve its efficiency, we synthesized and tested various LAH4 derivatives. The evaluation of their DNA delivery capacity, as well as their transfection efficiency, makes accessible valuable information about structure-function correlations. In particular, our results indicate the threshold peptide concentration for endosomal escape and conformational preferences for the peptide. Furthermore, the results indicate that a fine balance of peptide-DNA interactions is responsible for the high transfection activity of LAH4. Taken together, the data also suggest that the peptides efficiently transport DNA into the cytoplasm and that the creation of more potent transfection compounds probably needs improvement of other steps during the process.

Cell Line↗

PubMed2006

A high throughput method for genome-wide analysis of retroviral integration.

Retroviral and lentiviral vectors integrate their DNA into the host cell genome leading to stable transgene expression. Integration preferentially occurs in the proximity of active genes, and may in some case disturb their activity, with adverse toxic consequences. To efficiently analyze high numbers of lentiviral insertion sites in the DNA of transduced cells, we developed an improved high-throughput method called vector integration tag analysis (VITA). VITA is based on the identification of Genomic Tags associated to the insertion sites, which are used as signatures of the integration events. We use the capacity of MmeI to cleave DNA at a defined distance of its recognition site, in order to generate 21 bp long tags from libraries of junction fragments between vector and cellular DNA. The length of the tags is sufficient in most cases, to identify without ambiguity an unique position in the human genome. Concatenation, cloning and sequencing of the tags allow to obtain information about 20-25 insertion sites in a single sequencing reaction. As a validation of this method, we have characterized 1349 different lentiviral vector insertion sites in transduced HeLa cells, from only 487 sequencing reactions, with a background of <2% false positive tags.

Cell Line, Tumor↗

PubMed2006

Simple conditioning with monospecific CD4+CD25+ regulatory T cells for bone marrow engraftment and tolerance to multiple gene products.

A major impediment to gene replacement therapy is immune elimination of genetically modified cells. In principle, this can be dealt with by inducing a strong, specific, and enduring tolerance through engraftment of transgene-modified autologous bone marrow (BM). Because usual myeloablation and/or immunosuppression are risk factors in most pathologies, we assessed the potential of monospecific CD4(+)CD25(+) regulatory T cells (Tregs) to engraft minor-mismatched BM without preconditioning. We found that as few as 5 x 10(4) Tregs directed to the male DBY protein promote the engraftment of foreign male BM into sex-mismatched female hosts, establishing sustained chimerism in all hematopoeitic compartments. We achieved concomitantly strong tolerance to all foreign antigens expressed in the BM, likely occurring through induction of anergy and/or deletion of antidonor T cells. Chimerism was obtained in thymectomized mice too, underlining the major role of peripheral tolerance mechanisms in our system. This allowed us to engraft gene-modified tissues while preserving full immunocompetence to third-party antigens. Our results demonstrate that very few donor-specific Tregs are effective as the sole conditioning to induce mixed molecular chimerism and long-term tolerance to multiple foreign antigens.

Animals↗

PubMed2006

Dialysis-assisted two-dimensional gel electrophoresis.

2-DE is an important tool in proteomics research. However, intrinsic gel-to-gel variability of 2-DE often masks the biological differences between the samples and compromises quantitative comparison of protein expression levels. Here, we describe a modification of 2-DE that results in improved matching and quantification of proteins. This was accomplished by performing IEF of two samples in two IPG strips separated by a dialysis membrane. After IEF running, the strips were separated and the SDS-PAGE dimension was accomplished on two individual gels. After gel staining with CBB, ImageMaster 2D Platinum software (Amersham) was used for spot detection and quantification. Analysis of protein extracts from C2C12 myoblasts by this method resulted in 99% spot-matching efficiency and CV in stain intensity (% volume) was less than 0.5 for 98% of spots. We conclude that this technique, called dialysis-assisted gel electrophoresis, gives superior spot matching and quantitative reproducibility compared to IEF conducted on separate strips.

Dialysis↗

PubMed2006

Gene expression profiling to monitor therapeutic and adverse effects of antisense therapies for Duchenne muscular dystrophy.

OBJECTIVES: The objective of this study was to assess the utility of the gene expression profiling technique for the preclinical evaluation of drug efficacy and safety, taking a new therapeutic approach for Duchenne muscular dystrophy (DMD) as an example. METHODS: Muscles from dystrophin-deficient (mdx) mice, a well-characterized animal model for DMD, were injected with antisense constructs that restore the open reading frame in the Dmd gene. Synthetic antisense oligonucleotides (AONs) complexed with different carriers to enhance cellular uptake and recombinant adeno-associated virus (rAAV)-expressed antisense sequences were evaluated. Muscular gene expression profiles were analyzed on oligonucleotide microarrays. RESULTS: Polyethylenimine (PEI)-complexed AONs restored the reading frame slightly more effectively than uncomplexed, F127- or Optison-complexed AONs. However, PEI induced the expression of many immune genes, reflecting an aggravation of the inflammation present in untreated mdx mice. Expression profiles in Optison and F127-injected muscles were similar to those of saline treated muscles, implying that these carriers did not evoke adverse responses. Due to moderate levels of exon skipping, a significant shift toward wild-type expression levels was not detected. Injection with rAAV vectors resulted in much higher production of dystrophin and greatly improved the histological appearance of the muscle. Depending on the efficacy of the treatment, the expression of genes previously shown to be elevated in muscular dystrophies, partly or completely returned to wild-type expression levels. Reductions in inflammation and fibrosis were among the most prominent changes observed. CONCLUSION: Expression profiling is a powerful tool for the evaluation of both desired and adverse effects of new pharmacological therapies. It is sensitive and detects changes that are not histologically visible. In addition, its ability to simultaneously monitor a large number of different biological processes not only reduces the number of different assays required in preclinical research and clinical trials, but may also assist in the early detection of potential side effects.

Animals↗

PubMed2005

A partial down-regulation of WASP is sufficient to inhibit podosome formation in dendritic cells.

The Wiskott Aldrich syndrome protein (WASP) is a hematopoietic-specific cytoskeletal regulator that is necessary for induction of normal immunity. In the context of effective gene therapy for WAS, cellular models of human WASP deficiency are important for definition of the threshold of protein expression required for optimal activity. Using lentiviral vector-mediated RNA interference (RNAi), we were able to down-regulate the levels of human WASP in cell lines and primary cells. In dendritic cells (DC), RNAi-induced WASP deficiency did not impair phenotypic maturation but perturbed cytoskeletal organization. As a result, podosomes, which are actin-rich structures present in immature adherent DC, were formed less efficiently and motility was disturbed. Overall, treatment of cells with RNAi recapitulated the phenotype of cells derived from patients or animals with inactivating mutations of the WAS gene. Interestingly, reduction of the levels of WASP to about 60% of normal was sufficient to inhibit the formation of podosomes in DC, implying that this cell type requires near-normal levels of WASP to sustain physiological cytoskeleton-dependent activities.

Cell Movement↗

PubMed2005

The antibiotic and DNA-transfecting peptide LAH4 selectively associates with, and disorders, anionic lipids in mixed membranes.

The histidine-rich amphipathic peptide LAH4 has antibiotic and DNA delivery capabilities. The peptide has a strong affinity for anionic lipids found in the outer membrane of bacterial membranes. A role for anionic lipids in release of cationic plasmid-containing complexes has been proposed previously, and disruption of membrane asymmetry and presentation of phosphatidylserine (PS) in the membrane outer leaflet is a general feature observed in diseased mammalian cells. Therefore, to understand the peptide-lipid interactions in more detail, solid-state NMR experiments on model membranes have been performed. 31P MAS NMR on mixed phosphatidylcholine (PC)/PS and PC/phosphatidylglycerol (PG) membranes has been used to demonstrate a strong interaction between LAH4 and anionic lipids. By using deuterated lipids and wide-line 2H NMR when probing lipid chain order, it is demonstrated that LAH4 preferentially interacts with PS over PC and effectively disorders the anionic PS lipid fatty acyl chains. In addition, we demonstrate that the efficiency of gene transfer in vitro to different cell lines is closely related to the degree of disruption of PS acyl chains for four isomers of LAH4. This work suggests a mechanism of selective destabilization by LAH4 of anionic lipids in the membranes of cells during transfection with implications for nucleic acid delivery in vivo.

Amino Acid Sequence↗

PubMed2005

Sustained phenotypic reversion of junctional epidermolysis bullosa dog keratinocytes: Establishment of an immunocompetent animal model for cutaneous gene therapy.

Gene transfer represents the unique therapeutic issue for a number of inherited skin disorders including junctional epidermolysis bullosa (JEB), an untreatable genodermatose caused by mutations in the adhesion ligand laminin 5 (alpha3beta3gamma2) that is secreted in the extracellular matrix by the epidermal basal keratinocytes. Because gene therapy protocols require validation in animal models, we have phenotypically reverted by oncoretroviral transfer of the curative gene the keratinocytes isolated from dogs with a spontaneous form of JEB associated with a genetic mutation in the alpha3 chain of laminin 5. We show that the transduced dog JEB keratinocytes: (1) display a sustained secretion of laminin 5 in the extracellular matrix; (2) recover the adhesion, proliferation, and clonogenic capacity of wild-type keratinocytes; (3) generate fully differentiated stratified epithelia that after grafting on immunocompromised mice produce phenotypically normal skin and sustain permanent expression of the transgene. We validate an animal model that appears particularly suitable to demonstrate feasibility, efficacy, and safety of genetic therapeutic strategies for cutaneous disorders before undertaking human clinical trials.

Animals↗

PubMed2005

Safety and efficacy of AAV-mediated calpain 3 gene transfer in a mouse model of limb-girdle muscular dystrophy type 2A.

Calpainopathy (limb-girdle muscular dystrophy type 2A, LGMD2A) is a recessive muscular disorder caused by deficiency in the calcium-dependent cysteine protease calpain 3. To date, no treatment exists for this disease. We evaluated the potential of recombinant adeno-associated virus (rAAV) vectors for gene therapy in a murine model for LGMD2A. To drive the expression of calpain 3, we used rAAV2/1 pseudotyped vectors and muscle-specific promoters to avoid calpain 3 cell toxicity. We report efficient and stable transgene expression in muscle with restoration of the proteolytic activity and without evident toxicity. In addition, calpain 3 was correctly targeted to the sarcomere. Moreover, its presence resulted in improvement of the histological features and in therapeutic efficacy at the physiological levels, including correction of atrophy and full rescue of the contractile force deficits. Our results establish the feasibility of AAV-mediated calpain 3 gene transfer as a therapeutic approach.

Animals↗

PubMed2005

Efficient control of gene expression in the hematopoietic system using a single Tet-on inducible lentiviral vector.

This work addresses the problem of efficient control of gene expression in the context of viral vectors, which still represents a difficult challenge. A number of lentiviral vectors incorporating the different elements of regulatable transcriptional systems have been described, but they fail to perform satisfactorily either because of a poor dynamic range of transcription levels or because they display high background activities in the uninduced state and mediocre inducer response. We report here on the systematic comparison of vector designs containing the elements of the doxycycline-inducible Tet-on system in their most advanced versions (rtTA2S-M2 transactivator and tTS(Kid) repressor). We show that a simple "all-in-one" vector can be obtained and used for efficient control of transgene expression in long-term tissue culture and in the hematopoietic system of mice following bone marrow transplantation. Using this vector, the uninduced state can be kept at background levels and induction factors of 100-fold are repeatedly obtained over months both in tissue culture and in vivo. Interestingly, the low background activity of the all-in-one vector renders the use of the tTS repressor dispensable, avoiding the problem of progressive loss of inducibility over time associated with irreversible modifications of the chromatin surrounding proviral sequences.

Animals↗

PubMed2005

Cationic steroid antibiotics demonstrate DNA delivery properties.

Recently, cationic steroids have been developed that display broad-spectrum antibacterial activity. These compounds, characterized by the presence of several amino groups, present a facially amphiphilic morphology. Formulations containing such steroids were tested for their ability to facilitate the uptake of a reporter plasmid into various cell lines. The results show that, when associated with the naturally occurring zwitterionic lipid dioleoyl-phosphatidylethanolamine (DOPE), cationic steroid antibiotics allow for transfection levels comparable to those obtained with DOTAP. The activity of the amphiphilic mixture was nearly unaffected by bafilomycin A1 and chloroquine treatment, suggesting a mechanism that is independent of the acidification process associated with endocytosis. Collectively, our results show that DNA delivery agents possessing strong antibacterial properties can be obtained by conjugating amino groups to a steroid nucleus.

Anti-Bacterial Agents↗

PubMed2005

Long-term immune reconstitution in RAG-1-deficient mice treated by retroviral gene therapy: a balance between efficiency and toxicity.

Severe combined immunodeficiency (SCID) caused by mutations in RAG1 or RAG2 genes is characterized by a complete block in T- and B-cell development. The only curative treatment is allogeneic hematopoietic stem cell transplantation, which gives a high survival rate (90%) when an HLA-genoidentical donor exists but unsatisfactory results when only partially compatible donors are available. We have thus been interested in the development of a potential alternative treatment by using retroviral gene transfer of a normal copy of RAG1 cDNA. We show here that this approach applied to RAG-1-deficient mice restores normal B- and T-cell function even in the presence of a reduced number of mature B cells. The reconstitution is stable over time, attesting to a selective advantage of transduced progenitors. Notably, a high transgene copy number was detected in all lymphoid organs, and this was associated with a risk of lymphoproliferation as observed in one mouse. Altogether, these results demonstrate that correction of RAG-1 deficiency can be achieved by gene therapy in immunodeficient mice but that human application would require the use of self-inactivated vector to decrease the risk of lymphoproliferative diseases.

Animals↗

PubMed2005

Direct recognition by alphabeta cytolytic T cells of Hfe, a MHC class Ib molecule without antigen-presenting function.

Crystallographic analysis of human Hfe has documented an overall structure similar to classical (class Ia) MHC molecules with a peptide binding groove deprived of ligand. Thus, to address the question of whether alphabeta T cells could recognize MHC molecules independently of bound ligands, we studied human and mouse Hfe interactions with T lymphocytes. We provide formal evidence of direct cytolytic recognition of human Hfe by mouse alphabeta T cell receptors (TCR) in HLA-A*0201 transgenic mice and that this interaction results in ZAP-70 phosphorylation. Furthermore, direct recognition of mouse Hfe molecules by cytotoxic T lymphocytes (CTLs) was demonstrated in DBA/2 Hfe knockout mice. These CTLs express predominantly two T cell antigen receptor alpha variable gene segments (AV6.1 and AV6.6). Interestingly, in wild-type mice we identified a subset of CD8+ T cells positively selected by Hfe that expresses the AV6.1/AV6.6 gene segments. T cell antigen receptor recognition of MHC molecules independently of bound ligand has potential general implications in alloreactivity and identifies in the Hfe case a cognitive link supporting the concept that the immune system could be involved in the control of iron metabolism.

Animals↗

PubMed2005

Global transcriptional characterization of SP and MP cells from the myogenic C2C12 cell line: effect of FGF6.

With the use of Hoechst staining techniques, we have previously shown that the C2C12 myogenic cell line contains a side population (SP) that is largely increased in the presence of fibroblast growth factor 6 (FGF6). Here, we compared transcriptional profiles from SP and main population (MP) cells from either C2C12 or FGF6-expressing C2C12. Expression profiles of SPs show that these cells are less differentiated than MPs and display some similarities to stem cells. Moreover, principal component analysis made it possible to distinguish specific contributions of either FGF6 or differentiation effects on gene expression profiles. This demonstrated that FGF6-expanded SPs were similar to parental C2C12-derived SPs. Conversely, FGF6-treated MPs differed from parental MPs and were more related to SP cells. These results show that FGF6 pushed committed myogenic cells toward a more immature phenotype resulting in the accumulation of cells with a SP phenotype. We propose that FGF6 conditioning could provide a way to expand the pool of immature cells by myoblast dedifferentiation.

Animals↗

PubMed2005

Femoral intra-arterial injection: a tool to deliver and assess recombinant AAV constructs in rodents whole hind limb.

With the aim of simplifying recombinant-adeno-associated virus (rAAV) delivery in muscle, a new femoral intra-arterial technique was designed and tested in rodents (rats and mice). Two serotypes, several promoters and transgenes (reporter or therapeutic) were tested using this administration route. The new route is both easy to perform and efficient. Its usefulness as a tool to assess gene delivery constructs in the muscle was established in the context of recombinant AAV serotypes 1 and 2, and with the ubiquitous CMV and two muscle-specific (C5-12 and CK6) promoters. Both serum monitoring of a secreted protein (murine alkaline phosphatase: muSEAP) and slide staining were used to compare the different constructs. Significantly different patterns of expression in kinetics of expression (muSEAP) and homogeneity of fiber transduction (staining) were evidenced with the different promoters tested, and compared with intra-muscular expression patterns. Detailed studies of differential transduction in leg and thigh muscles showed equivalent efficacy, except in rectus femoris, and to a lesser extent in soleus. In light of these results and prior data, intra-arterially mediated gene transfer mechanism is discussed.

Alkaline Phosphatase↗