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ABSTRACT: Many genetic diseases are induced by mutations disturbing the maturation of pre-mRNAs, often affecting splicing. Antisense oligoribonucleotides (AONs) have been used to modulate splicing thereby circumventing the deleterious effects of mutations. Stable delivery of antisense sequences is achieved by linking them to small nuclear RNA (snRNAs) delivered by viral vectors, as illustrated by studies where therapeutic exon skipping was obtained in animal models of Duchenne muscular dystrophy (DMD). Yet, clinical translation of these approaches is limited by the amounts of vector to be administered. In this respect, maximizing the amount of snRNA antisense shuttle delivered by the vector is essential. Here, we have used a muscle- and heart-specific enhancer (MHCK) to drive the expression of U7 snRNA shuttles carrying antisense sequences against the human or murine DMD pre-mRNAs. Although antisense delivery and subsequent exon skipping were improved both in tissue culture and in vivo, we observed the formation of additional U7 snRNA by-products following gene transfer. These included aberrantly 3' processed as well as unprocessed species that may arise because of the saturation of the cellular processing capacity. Future efforts to increase the amounts of functional U7 shuttles delivered into a cell will have to take this limitation into account.
Molecular therapy. Nucleic acids. 01/2012; 1:e31.
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Glenn Dallérac,
Caroline Perronnet,
Carine Chagneau,
Pascale Leblanc-Veyrac,
Nathalie Samson-Desvignes,
Elise Peltekian, Olivier Danos,
Luis Garcia,
Serge Laroche,
Jean-Marie Billard,
Cyrille Vaillend
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ABSTRACT: Duchenne muscular dystrophy (DMD) is caused by the absence of dystrophin, a protein that fulfills important functions in both muscle and brain. The mdx mouse model of DMD, which also lacks dystrophin, shows a marked reduction in γ-aminobutyric acid type A (GABA(A))-receptor clustering in central inhibitory synapses and enhanced long-term potentiation (LTP) at CA3-CA1 synapses of the hippocampus. We have recently shown that U7 small nuclear RNAs modified to encode antisense sequences and expressed from recombinant adeno-associated viral (rAAV) vectors are able to induce skipping of the mutated exon 23 and to rescue expression of a functional dystrophin-like product both in the muscle and nervous tissue in vivo. In the brain, this rescue was accompanied by restoration of both the size and number of hippocampal GABA(A)-receptor clustering. Here, we report that 25.2±8% of re-expression two months after intrahippocampal injection of rAAV reverses the abnormally enhanced LTP phenotype at CA3-CA1 synapses of mdx mice. These results suggests that dystrophin expression indirectly influences synaptic plasticity through modulation of GABA(A)-receptor clustering and that re-expression of the otherwise deficient protein in the adult can significantly alleviate alteration of neural functions in DMD.
Neurobiology of Disease 05/2011; 43(3):635-41. · 5.40 Impact Factor
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ABSTRACT: Dystrophin, the cytoskeletal protein whose defect is responsible for Duchenne muscular dystrophy (DMD), is normally expressed in both muscles and brain. Genetic loss of brain dystrophin in the mdx mouse model of DMD reduces the capacity for type A gamma-aminobutyric acid (GABA(A))-receptor clustering in central inhibitory synapses, which is thought to be a main molecular defect leading to brain and cognitive alterations in this syndrome. U7 small nuclear RNAs modified to encode antisense sequences and expressed from recombinant adeno-associated viral (rAAV) vectors have proven efficient after intramuscular injection to induce skipping of the mutated exon 23 and rescue expression of a functional dystrophin-like product in muscle tissues of mdx mice in vivo. Here, we report that intrahippocampal injection of a single dose of rAAV2/1-U7 can rescue substantial levels of brain dystrophin expression (15-25%) in mdx mice for months. This is sufficient to completely restore GABA(A)-receptor clustering in pyramidal and dendritic layers of CA1 hippocampus, suggesting exon-skipping strategies offer the prospect to investigate and correct both brain and muscle alterations in DMD. This provides new evidence that in the adult brain dystrophin is critical for the control of GABA(A)-receptor clustering, which may have an important role in activity-dependent synaptic plasticity in hippocampal circuits.
Molecular Therapy 09/2010; 18(9):1683-8. · 6.87 Impact Factor
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ABSTRACT: Following entry, uncoating, and reverse transcription, a number of cellular proteins become associated with the human immunodeficiency virus type 1 (HIV-1) pre-integration complex (PIC). With the goal of obtaining reagents for the analysis of the HIV-1 PIC composition and localisation, we have constructed functional integrase (IN) and matrix (MA) proteins that can be biotinylated during virus production and captured using streptavidin-coated beads.
Although the labelled C-terminus allows for the sensitive detection of virion-associated IN, it becomes inaccessible in the presence of cellular proteins. This masking is not dependent on the nature of the tag and does not occur with the tagged MA. It was not observed either with an IN mutant unable to interact with LEDGF/p75, or when LEDGF/p75 was depleted from cells.
Our observation suggests that a structural rearrangement or oligomerization of the IN protein occurs during the early steps of infection and that this process is related to the presence of LEDGF/p75.
Retrovirology 04/2010; 7:27. · 6.47 Impact Factor
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Delphine Peric,
Christelle Durrant-Arico,
Christophe Delenda,
Thierry Dupré,
Pascale De Lonlay,
Hélène Ogier de Baulny,
Cécile Pelatan,
Brigitte Bader-Meunier, Olivier Danos,
Isabelle Chantret,
Stuart E H Moore
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ABSTRACT: Biosynthesis of the dolichol linked oligosaccharide (DLO) required for protein N-glycosylation starts on the cytoplasmic face of the ER to give Man(5)GlcNAc(2)-PP-dolichol, which then flips into the ER for further glycosylation yielding mature DLO (Glc(3)Man(9)GlcNAc(2)-PP-dolichol). After transfer of Glc(3)Man(9)GlcNAc(2) onto protein, dolichol-PP is recycled to dolichol-P and reused for DLO biosynthesis. Because de novo dolichol synthesis is slow, dolichol recycling is rate limiting for protein glycosylation. Immature DLO intermediates may also be recycled by pyrophosphatase-mediated cleavage to yield dolichol-P and phosphorylated oligosaccharides (fOSGN2-P). Here, we examine fOSGN2-P generation in cells from patients with type I Congenital Disorders of Glycosylation (CDG I) in which defects in the dolichol cycle cause accumulation of immature DLO intermediates and protein hypoglycosylation.
In EBV-transformed lymphoblastoid cells from CDG I patients and normal subjects a correlation exists between the quantities of metabolically radiolabeled fOSGN2-P and truncated DLO intermediates only when these two classes of compounds possess 7 or less hexose residues. Larger fOSGN2-P were difficult to detect despite an abundance of more fully mannosylated and glucosylated DLO. When CDG Ig cells, which accumulate Man(7)GlcNAc(2)-PP-dolichol, are permeabilised so that vesicular transport and protein synthesis are abolished, the DLO pool required for Man(7)GlcNAc(2)-P generation could be depleted by adding exogenous glycosylation acceptor peptide. Under conditions where a glycotripeptide and neutral free oligosaccharides remain predominantly in the lumen of the ER, Man(7)GlcNAc(2)-P appears in the cytosol without detectable generation of ER luminal Man(7)GlcNAc(2)-P.
The DLO pools required for N-glycosylation and fOSGN2-P generation are functionally linked and this substantiates the hypothesis that pyrophosphatase-mediated cleavage of DLO intermediates yields recyclable dolichol-P. The kinetics of cytosolic fOSGN2-P generation from a luminally-generated DLO intermediate demonstrate the presence of a previously undetected ER-to-cytosol translocation process for either fOSGN2-P or DLO.
PLoS ONE 01/2010; 5(7):e11675. · 4.09 Impact Factor
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ABSTRACT: Foxp3(+) Treg are crucial for the maintenance of self-tolerance and have been shown to control CD8(+) T-cell effector functions. In addition, Treg are thought to control the priming of CD8(+) T cells, which recognize the same antigens as Treg. Taking advantage of our model of peripheral tolerance induction to influenza hemagglutinin (HA) after HA gene transfer, we found that HA-specific Treg suppress antigen-linked CTL responses through early blockade of CD8(+) T-cell expansion. Confronted with their cognate antigen, Treg expand more rapidly than CD8(+) T cells and are highly suppressive only during the initial stages of immune priming. They nullify HA-specific CD8(+) T-cell responses, local inflammatory responses and rejection of HA transduced cells. When HA gene transfer is performed with extensive tissue inflammation, HA-specific Treg are less effective but still reduce the frequency of newly primed HA-specific CD8(+) T cells and the ensuing frequency of memory CD8(+) T cells. Our results demonstrate that Treg control CTL priming in an antigen-specific manner at the level of T-cell expansion, highlighting how self-reactive Treg could prevent the induction of autoimmune responses through selective blockade of autoreactive T-cell proliferation.
European Journal of Immunology 10/2009; 40(2):339-50. · 5.10 Impact Factor
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ABSTRACT: Among the integrative gene therapy vectors developed to date, human immunodeficiency virus type 1 (HIV-1)-derived lentiviral vectors (LV) are distinguished by their capacity to infect both dividing and non-dividing cells. Recombinant LV particles contain viral proteins necessary for their packaging, infectious and integrating functions. Like the parental HIV-1 virus they are able to acquire various cellular proteins, but the number and localisation of these proteins are poorly characterised. In the present study we used 2-DE followed by MALDI-TOF to quantify the protein content of several types of vesicular stomatitis virus G-pseudotyped LV including those that were extensively purified in the perspective of clinical gene therapy studies. A proteinase K treatment was used to distinguish between cellular proteins incorporated into virions (I-proteins) and those co-purified with vectors (C-proteins). We found 10 C-proteins and 18 I-proteins associated with LV. Copy numbers for these core I-proteins varied from 5 (AIP-1/ALIX) to 280 (Cyclophilin A) per vector particle. Three novel I-proteins, guanine nucleotide-binding protein 2, L-lactate dehydrogenase B chain and hnRNP core protein A1, were found. This study defines for the first time, the protein stoichiometry of infectious HIV-1-derived LV particles.
Proteomics 08/2009; 9(14):3666-76. · 4.43 Impact Factor
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ABSTRACT: The genomic integration of a lentiviral vector developed for the treatment of Wiskott-Aldrich syndrome (WAS) was assessed by localizing the vector insertion sites (IS) in a murine model of gene therapy for the disease.
Transduced hematopoietic progenitor cells were transplanted into mice or cultured in vitro. The IS were determined in the genomic DNA from blood, the bone marrow of the animals and from cultured cells.
Sequencing vector-genomic DNA junctions yielded more than 150 IS of which 50-70% were located in transcription units. To obtain additional sequences from the population of cultured cells, we used a vector-tag concatenation technique providing 190 additional IS. Altogether, the profiles confirmed the bias for integration in transcription units. The vector did not congregate as hotspots and did not appear to target specific categories of genes. The diversity of the IS reflected the initial marking of a polyclonal population of cells. However, relatively few vector IS were found in vivo because only 30-40 unique IS were identified in each mouse using this approach. Although four to ten IS were shared by the blood and bone marrow, no common IS was found between mice or between any mouse and the cultured cells.
Taken as a whole, the pattern of genomic insertion of the WAS lentiviral vector was diverse and similar to that previously described for other HIV-1-derived lentiviral vectors. Testing cells destined for transplantation is unlikely to predict specific IS to be selected in vivo.
The Journal of Gene Medicine 06/2009; 11(8):645-54. · 2.48 Impact Factor
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ABSTRACT: Current strategies for gene therapy of inherited diseases consist in adding functional copies of the gene that is defective. An attractive alternative to these approaches would be to correct the endogenous mutated gene in the affected individual. This study presents a quantitative comparison of the repair efficiency using different forms of donor nucleic acids, including synthetic DNA oligonucleotides, double stranded DNA fragments with sizes ranging from 200 to 2200 bp and sequences carried by a recombinant adeno-associated virus (rAAV-1). Evaluation of each gene repair strategy was carried out using two different reporter systems, a mutated eGFP gene or a dual construct with a functional eGFP and an inactive luciferase gene, in several different cell systems. Gene targeting events were scored either following transient co-transfection of reporter plasmids and donor DNAs, or in a system where a reporter construct was stably integrated into the chromosome.
In both episomal and chromosomal assays, DNA fragments were more efficient at gene repair than oligonucleotides or rAAV-1. Furthermore, the gene targeting frequency could be significantly increased by using DNA repair stimulating drugs such as doxorubicin and phleomycin.
Our results show that it is possible to obtain repair frequencies of 1% of the transfected cell population under optimized transfection protocols when cells were pretreated with phleomycin using rAAV-1 and dsDNA fragments.
BMC Biotechnology 05/2009; 9:35. · 2.35 Impact Factor
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Anna Buj-Bello,
Françoise Fougerousse,
Yannick Schwab,
Nadia Messaddeq,
Danièle Spehner,
Christopher R Pierson,
Muriel Durand,
Christine Kretz, Olivier Danos,
Anne-Marie Douar,
Alan H Beggs,
Patrick Schultz,
Marie Montus,
Patrice Denèfle,
Jean-Louis Mandel
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ABSTRACT: Myotubular myopathy (XLMTM, OMIM 310400) is a severe congenital muscular disease due to mutations in the myotubularin gene (MTM1) and characterized by the presence of small myofibers with frequent occurrence of central nuclei. Myotubularin is a ubiquitously expressed phosphoinositide phosphatase with a muscle-specific role in man and mouse that is poorly understood. No specific treatment exists to date for patients with myotubular myopathy. We have constructed an adeno-associated virus (AAV) vector expressing myotubularin in order to test its therapeutic potential in a XLMTM mouse model. We show that a single intramuscular injection of this vector in symptomatic Mtm1-deficient mice ameliorates the pathological phenotype in the targeted muscle. Myotubularin replacement in mice largely corrects nuclei and mitochondria positioning in myofibers and leads to a strong increase in muscle volume and recovery of the contractile force. In addition, we used this AAV vector to overexpress myotubularin in wild-type skeletal muscle and get insight into its localization and function. We show that a substantial proportion of myotubularin associates with the sarcolemma and I band, including triads. Myotubularin overexpression in muscle induces the accumulation of packed membrane saccules and presence of vacuoles that contain markers of sarcolemma and T-tubules, suggesting that myotubularin is involved in plasma membrane homeostasis of myofibers. This study provides a proof-of-principle that local delivery of an AAV vector expressing myotubularin can improve the motor capacities of XLMTM muscle and represents a novel approach to study myotubularin function in skeletal muscle.
Human Molecular Genetics 08/2008; 17(14):2132-43. · 7.64 Impact Factor
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Marc Bartoli,
Evelyne Gicquel,
Laetitia Barrault,
Tayebeh Soheili,
Marie Malissen,
Bernard Malissen,
Nathalie Vincent-Lacaze,
Norma Perez,
Bjarne Udd, Olivier Danos,
Isabelle Richard
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ABSTRACT: Limb girdle muscular dystrophy type 2D (LGMD2D, OMIM600119) is a genetic progressive myopathy that is caused by mutations in the human alpha-sarcoglycan gene (SGCA). Here, we have introduced in mice the most prevalent LGMD2D mutation, R77C. It should be noted that the natural murine residue at this position is a histidine. The model is, therefore, referred as Sgca(H77C/H77C). Unexpectedly, we observed an absence of LGMD2D-like phenotype at histological or physiological level. Using a heterologous cellular model of the sarcoglycan complex formation, we showed that the R77C allele encodes a protein that fails to be delivered to its proper cellular localization in the plasma membrane, and consequently to the disappearance of a positively charged residue. Subsequently, we transferred an AAV vector coding for the human R77C protein in the muscle of Sgca-null mice and were able to pharmacologically rescue the R77C protein from endoplasmic reticulum-retention using proteasome or mannosidase I inhibitors. This suggests a therapeutic approach for LGMD2D patients carrying mutations that impair alpha-sarcoglycan trafficking.
Human Molecular Genetics 06/2008; 17(9):1214-21. · 7.64 Impact Factor
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ABSTRACT: Exon-skipping AAV1-U7-associated therapy is a promising treatment for Duchenne muscular dystrophy (DMD). We have shown earlier that the newly rescued dystrophin protein is stably expressed for months in mice and dogs, and does not induce immune rejection of transduced fibers. In this study, we used the dystrophic mdx mouse as a preclinical model to characterize the immune response to the adeno-associated virus 1 (AAV1) vector, and tested the feasibility of administering multiple AAV1 injections to extend the treatment to several muscles. We found that re-injections of AAV1 vector are compromised as early as 3 days after the first injection, coincident with a rapid increase in AAV1-specific immunoglobulin M (IgM) and IgG in the serum. Adoptive transfer of immune sera confirmed the rapid appearance of an AAV1 neutralization activity, and experiments with immunoglobulin-deficient (microKO) mice proved that antibodies (Abs) are the only effectors responsible for AAV1-U7 elimination. It is important to note, however, that the AAV2 vector still generated an adverse immune response in microKO mice. By blocking the T-B crosstalk with anti-CD40 Abs and CTLA4/Fc fusion protein, we found that a mere 5 days of immunomodulation treatment was sufficient to totally abrogate the formation of anti-AAV1 Abs and to allow for the correction of muscular dystrophy in multiple muscles, provided the treatment was administered during each challenge.
Molecular Therapy 04/2008; 16(3):541-7. · 6.87 Impact Factor
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ABSTRACT: Foxp3+ regulatory T cells (Tregs) play a pivotal role in the maintenance of peripheral T cell tolerance and are thought to interact with dendritic cells (DC) in secondary lymphoid organs. We analyzed here the in vivo requirements for selective expansion of Ag-specific Treg vs CD4+CD25- effector T cells and engagement of Ag-specific Treg-DC interactions in secondary lymphoid organs. Using i.v. Ag delivery in the absence of inflammation, we found that CD4+CD25+Foxp3+ Tregs undergo vigorous expansion and accumulate whereas naive CD4+CD25-Foxp3- T cells undergo abortive activation. Quantifying directly the interactions between Tregs and CD11c+ DC, we found that Tregs establish cognate contacts with endogenous CD11c+ DC in spleen and lymph nodes at an early time point preceding their expansion. Importantly, we observed that as few as 10(3) Tregs selectively expanded by i.v. Ag injection are able to suppress B and T cell immune responses in mouse recipients challenged with the Ag. Our results demonstrate that Tregs are selectively mobilized by Ag recognition in the absence of inflammatory signals, and can induce thereafter potent tolerance to defined Ag targets.
The Journal of Immunology 02/2008; 180(1):327-34. · 5.79 Impact Factor
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ABSTRACT: In the present study, modifications in cytosolic expressed proteins during human myoblast differentiation were studied by dialysis-assisted 2-DE (DAGE, [1]). About 1000 spots were analysed on the 5th and 13th day of differentiation with a dynamic range of protein expression exceeding 1000-fold. During myogenic differentiation, the number of nonmatching spots as well as the extent of quantitative differences between matched spots significantly increased. Over one hundred differentially expressed spots were excised and identified by MALDI-TOF MS. The differentiation-associated expression pattern of eight proteins was validated by Western blot analysis. Differential expression of several proteins was demonstrated for the first time in human myotubes. Interestingly, Ingenuity pathway analysis grouped 30 of these proteins into two overlapping networks containing as principal nodes IGF-1 and tumour necrosis factor, two proteins known to play a crucial role in cytogenesis. Our results illustrate the large rearrangement of the proteome during the differentiation of human myoblasts and provide evidence for new partners involved in this complex process.
PROTEOMICS 02/2008; 8(2):264-78. · 4.51 Impact Factor
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ABSTRACT: Exon-skipping AAV1-U7-associated therapy is a promising treatment for Duchenne muscular dystrophy (DMD). We have shown earlier that the newly rescued dystrophin protein is stably expressed for months in mice and dogs, and does not induce immune rejection of transduced fibers. In this study, we used the dystrophic mdx mouse as a preclinical model to characterize the immune response to the adeno-associated virus 1 (AAV1) vector, and tested the feasibility of administering multiple AAV1 injections to extend the treatment to several muscles. We found that re-injections of AAV1 vector are compromised as early as 3 days after the first injection, coincident with a rapid increase in AAV1-specific immunoglobulin M (IgM) and IgG in the serum. Adoptive transfer of immune sera confirmed the rapid appearance of an AAV1 neutralization activity, and experiments with immunoglobulin-deficient (μKO) mice proved that antibodies (Abs) are the only effectors responsible for AAV1-U7 elimination. It is important to note, however, that the AAV2 vector still generated an adverse immune response in μKO mice. By blocking the T-B crosstalk with anti-CD40 Abs and CTLA4/Fc fusion protein, we found that a mere 5 days of immunomodulation treatment was sufficient to totally abrogate the formation of anti-AAV1 Abs and to allow for the correction of muscular dystrophy in multiple muscles, provided the treatment was administered during each challenge.
Molecular Therapy 01/2008; 16(3):541-547. · 6.87 Impact Factor
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ABSTRACT: Stable and safe corrective gene transfer in stem keratinocytes is necessary for ensuring success in cutaneous gene therapy. There have been numerous encouraging preclinical approaches to cutaneous gene therapy in the past decade, but it is only recently that a human volunteer suffering from junctional epidermolysis bullosa could be successfully grafted using his own non-selected, genetically corrected epidermal keratinocytes. However, ex vivo correction of cancer-prone genetic disorders necessitates a totally pure population of stably transduced stem keratinocytes for grafting. Antibiotic selection is not compatible with the need for full respect for natural cell fate potential and avoidance of immunogenic response in vivo. In order to surmount these problems, we developed a strategy for selecting genetically modified stem cell keratinocytes. Driving ectopic expression of CD24 (a marker of post-mitotic keratinocytes) at the surface of clonogenic keratinocytes permitted their full selection. Engineered keratinocytes expressing CD24 and the green fluorescent protein (GFP) tracer gene were shown to retain their original growth and differentiation potentials both in vitro and in vivo over 300 generations. Also, they did not exhibit signs of genetic instability. Using ectopic expression of CD24 as a selective marker of genetically modified human epidermal stem cells appears to be the first realistic approach to safe cutaneous gene therapy in cancer-prone disease conditions.
Molecular Therapy 01/2008; 15(12):2186-93. · 6.87 Impact Factor
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David Israeli,
Simindokht Ziaei,
Bernard Gjata,
Rachid Benchaouir,
Philippe Rameau,
Thibaut Marais,
So-Ichiro Fukada,
Masashi Segawa,
Hiroshi Yamamoto,
Patrick Gonin, Olivier Danos,
Luis Garcia
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ABSTRACT: The mouse mdr1a and mdr1b genes are expressed in skeletal muscle, though their precise role in muscle is unknown. Dystrophic muscle is characterized by repeated cycles of degeneration and regeneration. To explore the role of the mdr1 genes during muscle regeneration, we have created a triple knockout mouse lacking the mdr1a, mdr1b, and the dystrophin genes. The resulting ReX mice developed normally and were fertile. However, as adults, ReX had a higher proportion of degenerating muscle fibers and greater long-term loss of muscle mass than mdx. ReX muscles were also characterized by a reduced proportion of muscle side population (mSP) cells, of myogenic cells, and a reduced capacity for muscle regeneration. We found too that mSP cells derived from dystrophic muscle are more myogenic than those from normal muscle. Thus, in dystrophic muscle, the mdr1 gene plays an important role in the preservation of the mSP and of the myogenic regenerative potential. Moreover, our results suggest a hitherto unappreciated role of mdr1 in precursor cells of regenerating tissue; they therefore provide an important clue to the physiological significance of mdr1 expression in stem cells.
Experimental Cell Research 08/2007; 313(11):2438-50. · 3.58 Impact Factor
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ABSTRACT: We recently showed that the antibacterial histidine rich amphipathic peptide LAH4 has significant DNA transfection capabilities in the absence of serum. To further understand the transfection process and to develop the peptides for future applications, we have combined a range of biochemical and biophysical techniques, including fluorescence assisted cell sorting and (2)H solid-state NMR, to characterise the initial binding of the peptide/DNA complexes to the cell surface and the subsequent release of the complexes from the endosome in the presence of serum. Our results show that both primary and secondary peptide structure play important roles in both of these processes. Specifically, we show that an ideal helix length and positioning of the histidine residues should be maintained to obtain optimal resistance to serum effects and release of DNA from the endosome. Inclusion of d-amino acids at the peptide termini does not reduce serum effects however further enrichment of the peptides with histidine residues can enhance transfection efficiency in the presence of serum. The detailed understanding of these two key stages in the transfection process shows that LAH4-L1 and its derivatives are likely to be highly efficient and robust vectors for a range of applications.
Journal of Controlled Release 04/2007; 118(1):95-104. · 5.73 Impact Factor
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ABSTRACT: 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.
Journal of Molecular Medicine 03/2007; 85(2):191-201. · 4.67 Impact Factor
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Christophe Béroud,
Sylvie Tuffery-Giraud,
Masafumi Matsuo,
Dalil Hamroun,
Véronique Humbertclaude,
Nicole Monnier,
Marie-Pierre Moizard,
Marie-Antoinette Voelckel,
Laurence Michel Calemard,
Pierre Boisseau,
Martine Blayau,
Christophe Philippe,
Mireille Cossée,
Michel Pagès,
François Rivier, Olivier Danos,
Luis Garcia,
Mireille Claustres
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ABSTRACT: 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.
Human Mutation 03/2007; 28(2):196-202. · 5.69 Impact Factor
