Codon swapping of zinc finger nucleases confers expression in primary cells and in vivo from a single lentiviral vector

BACKGROUND: Zinc finger nucleases (ZFNs) are promising tools for genome editing for biotechnological as well as therapeutic purposes. Delivery remains a major issue impeding targeted genome modification. Lentiviral vectors are highly efficient for delivering transgenes into cell lines, primary cells and into organs, such as the liver. However, the reverse transcription of lentiviral vectors leads to recombination of homologous sequences, as found between and within ZFN monomers. METHODS: We used a codon swapping strategy to both drastically disrupt sequence identity between ZFN monomers and to reduce sequence repeats within a monomer sequence. We constructed lentiviral vectors encoding codon-swapped ZFNs or unmodified ZFNs from a single mRNA transcript. Cell lines, primary hepatocytes and newborn rats were used to evaluate the efficacy of integrative-competent (ICLV) and integrative-deficient (IDLV) lentiviral vectors to deliver ZFNs into target cells. RESULTS: We reduced total identity between ZFN monomers from 90.9% to 61.4% and showed that a single ICLV allowed efficient expression of functional ZFNs targeting the rat UGT1A1 gene after codon-swapping, leading to much higher ZFN activity in cell lines (up to 7-fold increase compared to unmodified ZFNs and 60% activity in C6 cells), as compared to plasmid transfection or a single ICLV encoding unmodified ZFN monomers. Off-target analysis located several active sites for the 5-finger UGT1A1-ZFNs. Furthermore, we reported for the first time successful ZFN-induced targeted DNA double-strand breaks in primary cells (hepatocytes) and in vivo (liver) after delivery of a single IDLV encoding two ZFNs. CONCLUSION: These results demonstrate that a codon-swapping approach allowed a single lentiviral vector to efficiently express ZFNs and should stimulate the use of this viral platform for ZFN-mediated genome editing of primary cells, for both ex vivo or in vivo applications

Cell therapy of burns

Severe burns remain a life threatening local and general inflammatory disease with often heavy sequelae, despite remarkable progress in their treatment in the last three decades. Cultured epidermal autografts, the first and still up-to-date cell therapy of burns, played a key role in that progress, and their drawbacks should be reduced with cultured dermal-epidermal substitutes. This review focuses on what could be the next major breakthrough in cell therapy of burns: mesenchymal stromal cells (MSCs). After summarizing current knowledge, including our own clinical experience, about MSCs in the pioneer field of cell therapy in radiation-induced burns, we discuss the strong rationale supporting the potential interest of MSCs in the treatment of thermal burns, including limited but promising pre-clinical and clinical data in wound healing and other acute inflammatory diseases. © 2012 The authors and IOS Press. All rights reserved

Cell therapy of burns

Severe burns remain a life-threatening local and general inflammatory condition often with serious sequelae, despite remarkable progress in their treatment over the past three decades. Cultured epidermal autografts, the first and still most up-to-date cell therapy for burns, plays a key role in that progress, but drawbacks to this need to be reduced by using cultured dermal-epidermal substitutes. This review focuses on what could be, in our view, the next major breakthrough in cell therapy of burns - use of mesenchymal stromal cells (MSCs). After summarizing current knowledge, including our own clinical experience with MSCs in the pioneering field of cell therapy of radiation-induced burns, we discuss the strong rationale supporting potential interest in MSCs in treatment of thermal burns, including limited but promising pre-clinical and clinical data in wound healing and acute inflammatory conditions other than burns. Practical options for future therapeutic applications of MSCs for burns treatment, are finally considered. © 2011 Blackwell Publishing Ltd

Polysaccharide-producing microalgae

SIGLEAvailable from CEN Saclay, Service de Documentation, 91191 - Gif-sur-Yvette Cedex (France) / INIST-CNRS - Institut de l'Information Scientifique et TechniqueFRFranc

Immobilisation de microalgues et productions exocellulaires

SIGLEAvailable from CEN Saclay, Service de Documentation, 91191 - Gif-sur-Yvette Cedex (France) / INIST-CNRS - Institut de l'Information Scientifique et TechniqueFRFranc

Agonist anti-ChemR23 mAb reduces tissue neutrophil accumulation and triggers chronic inflammation resolution

International audienceResolution of inflammation is elicited by proresolving lipids, which activate GPCRs to induce neutrophil apoptosis, reduce neutrophil tissue recruitment, and promote macrophage efferocytosis. Transcriptional analyses in up to 300 patients with Inflammatory Bowel Disease (IBD) identified potential therapeutic targets mediating chronic inflammation. We found that ChemR23, a GPCR targeted by resolvin E1, is overexpressed in inflamed colon tissues of severe IBD patients unresponsive to anti-TNF alpha or anti-alpha 4 beta 7 therapies and associated with significant mucosal neutrophil accumulation. We also identified an anti-ChemR23 agonist antibody that induces receptor signaling, promotes macrophage efferocytosis, and reduces neutrophil apoptosis at the site of inflammation. This ChemR23 mAb accelerated acute inflammation resolution and triggered resolution in ongoing chronic colitis models, with a significant decrease in tissue lesions, fibrosis and inflammation-driven tumors. Our findings suggest that failure of current IBD therapies may be associated with neutrophil infiltration and that ChemR23 is a promising therapeutic target for chronic inflammation

Effect of temperature, light intensity and dilution rate on the cellular composition of red alga Porphyridium cruentum in light-limited chemostat cultures

10.1007/BF01301953MIRCEN Journal of Applied Microbiology and Biotechnology42231-23