Quantification of lentiviral vector copy numbers in individual hematopoietic colony-forming cells shows vector dose-dependent effects on the frequency and level of transduction

Lentiviral vectors are effective tools for gene transfer and integrate variable numbers of proviral DNA copies in variable proportions of cells. The levels of transduction of a cellular population may therefore depend upon experimental parameters affecting the frequency and/or the distribution of vector integration events in this population. Such analysis would require measuring vector copy numbers (VCN) in individual cells. To evaluate the transduction of hematopoietic progenitor cells at the single-cell level, we measured VCN in individual colony-forming cell (CFC) units, using an adapted quantitative PCR (Q-PCR) method. The feasibility, reproducibility and sensitivity of this approach were tested with characterized cell lines carrying known numbers of vector integration. The method was validated by correlating data in CFC with gene expression or with calculated values, and was found to slightly underestimate VCN. In spite of this, such Q-PCR on CFC was useful to compare transduction levels with different infection protocols and different vectors. Increasing the vector concentration and re-iterating the infection were two different strategies that improved transduction by increasing the frequency of transduced progenitor cells. Repeated infection also augmented the number of integrated copies and the magnitude of this effect seemed to depend on the vector preparation. Thus, the distribution of VCN in hematopoietic colonies may depend upon experimental conditions including features of vectors. This should be carefully evaluated in the context of ex vivo hematopoietic gene therapy studies

Garnet lherzolite and garnet-spinel mylonite in the Ronda peridotite: Vestiges of Oligocene backarc mantle lithospheric extension in the western Mediterranean

International audienceUplift and exhumation of vast exposures of diamond facies, subcontinental mantle peridotite in the Western Mediterranean arc are attributed to tectonic scenarios including pure extension, transpression or subduction followed by delamination-driven or rollback-driven stretching. In the Ronda peridotite (southern Spain) the strong overprint of low-pressure assemblages has precluded accurate determination of the pressure and temperature conditions for the onset of exhumation that formed the spinel tectonite and garnet-spinel mylonite domain in this massif. Here we report unequivocal petrographic evidence for the existence of prekinematic, coarse-grained garnet lherzolite assemblages from the garnet-spinel mylonite domain of the Ronda peridotite. Application of well-calibrated geothermobarometers yields prekinematic minimum equilibration conditions of 2.4-2.7 GPa and 1020-1100 degrees C, demonstrating that the Ronda peridotite equilibrated at similar to 85 km depth before shearing. We also show the existence of synkinematic garnet and spinel assemblages that overprinted garnet lherzolite assemblages at 800-900 degrees C and 1.95-2.00 GPa. The decompressional cooling path and high pressure recorded by garnet-spinel mylonites rule out their formation by near-isobaric cooling above a subduction-collision wedge or during or after the emplacement of the peridotite massif into the crust. Ronda garnet-spinel mylonites represent the vestiges of subcontinental mantle ductile shear zones formed at early stages of lithosphere extension during backarc extension in the western Mediterranean. Southward to westward retreat of the African slab during the Oligocene-Early Miocene accounts for intense backarc lithosphere extension and development of the Ronda extensional shear zone, coeval with extreme thinning of the Alboran domain overlying crust

Deformation and Exhumation of the sub-Continental Mantle: Insigth from the Ronda Peridotite (Spain)

1 p.International audienc

Unequivocal garnet and garnet-spinel lherzolite assmeblages in the Ronda perodotite (S. Spain) and their implications for exhumation of subcontinental mantle in the Alboran basin

International audienc

AAV genome loss from dystrophic mouse muscles during AAV-U7 snRNA-mediated exon-skipping therapy

In the context of future adeno-associated viral (AAV)-based clinical trials for Duchenne myopathy, AAV genome fate in dystrophic muscles is of importance considering the viral capsid immunogenicity that prohibits recurring treatments. We showed that AAV genomes encoding non-therapeutic U7 were lost from mdx dystrophic muscles within 3 weeks after intramuscular injection. In contrast, AAV genomes encoding U7ex23 restoring expression of a slightly shortened dystrophin were maintained endorsing that the arrest of the dystrophic process is crucial for maintaining viral genomes in transduced fibers. Indeed, muscles treated with low doses of AAV-U7ex23, resulting in sub-optimal exon skipping, displayed much lower titers of viral genomes, showing that sub-optimal dystrophin restoration does not prevent AAV genome loss. We also followed therapeutic viral genomes in severe dystrophic dKO mice over time after systemic treatment with scAAV9-U7ex23. Dystrophin restoration decreased significantly between 3 and 12 months in various skeletal muscles, which was correlated with important viral genome loss, except in the heart. Altogether, these data show that the success of future AAV-U7 therapy for Duchenne patients would require optimal doses of AAV-U7 to induce substantial levels of dystrophin to stabilize the treated fibers and maintain the long lasting effect of the treatment. © The American Society of Gene and Cell Therapy

Actin scaffolding by clathrin heavy chain is required for skeletal muscle sarcomere organization

The ubiquitous clathrin heavy chain (CHC), the main component of clathrin-coated vesicles, is well characterized for its role in intracellular membrane traffic and endocytosis from the plasma membrane (PM). Here, we demonstrate that in skeletal muscle CH