Identification of Genes Regulating Gene Targeting by a High-Throughput Screening Approach

Homologous gene targeting (HGT) is a precise but inefficient process for genome engineering. Several methods for increasing its efficiency have been developed, including the use of rare cutting endonucleases. However, there is still room for improvement, as even nuclease-induced HGT may vary in efficiency as a function of the nuclease, target site, and cell type considered. We have developed a high-throughput screening assay for the identification of factors stimulating meganuclease-induced HGT. We used this assay to explore a collection of siRNAs targeting 19,121 human genes. At the end of secondary screening, we had identified 64 genes for which knockdown affected nuclease-induced HGT. Two of the strongest candidates were characterized further. We showed that siRNAs directed against the ATF7IP gene, encoding a protein involved in chromatin remodeling, stimulated HGT by a factor of three to eight, at various loci and in different cell types. This method thus led to the identification of a number of genes, the manipulation of which might increase rates of targeted recombination

The Compartmentalisation of Phosphorylated Free Oligosaccharides in Cells from a CDG Ig Patient Reveals a Novel ER-to-Cytosol Translocation Process

BACKGROUND: 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. METHODS AND PRINCIPAL FINDINGS: 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. CONCLUSIONS AND SIGNIFICANCE: 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

Normal cellular replication of Sendai virus without the trans-frame, nonstructural V protein

The Sendai virus V protein is a nonstructural trans-frame protein in which a highly conserved cys-rich Zn2+-binding domain is fused to the N-terminal half of the P protein via mRNA editing. Using a recently developed system in which infectious virus is recovered from cDNA, we have engineered a virus in which a translation stop codon was placed at the beginning of the V ORF. Translation of the V(stop) mRNA yields a W-like protein, i.e., a protein composed of the N-terminal half of the P protein alone which is naturally expressed at low levels from the P gene. This V-minus but W-augmented virus was found to replicate normally in cell culture and embryonated chicken eggs. The Sendai virus V protein is thus an accessory protein, and the cys-rich Zn2+-binding domain is likely to function in a specialized role during virus propagation

Développement des CAR-T dans les tumeurs solides

International audienceLes lymphocytes T à récepteur antigénique chimérique (CAR-T) ont démontré une efficacité remarquable dans certaines hémopathies malignes, notamment la leucémie aiguë lymphoblastique B et les lymphomes B pour les CAR anti-CD19. Les données dans les tumeurs solides restent quant à elles plutôt décevantes avec tout au plus quelques stabilisations et de rares réponses objectives observées. Ce manque d’efficacité peut être expliqué par différents facteurs : absence de cible spécifique (et donc risque accru de toxicité) et hétérogénéité d’expression, microenvironnement immunosuppresseur, homing insuffisant et faible pénétration intra-tumorale, défaut de persistance des CAR-T. De nombreuses approches sont actuellement envisagées pour contrecarrer ces différents mécanismes de résistance : construction de CAR bispécifiques ou utilisation de gates logiques, combinaison avec des inhibiteurs d’immune checkpoint ou CAR-T rendus insensibles aux molécules immunosuppressives, ajout de récepteurs de chémokines ou d’enzyme, combinaison avec un virus oncolytique, administration intra-tumorale, sélection de sous-populations mémoires et développement de CAR-T « armés » sécrétant des cytokines type IL-12, -15 ou -18. L’élaboration de CAR-T de dernière génération optimisés devrait à terme permettre de répondre à un besoin thérapeutique majeur notamment dans les tumeurs immunologiquement froides et/ou n’exprimant pas les molécules de CMH de classe I

The Versatility of Paramyxovirus RNA Polymerase Stuttering

Paramyxoviruses cotranscriptionally edit their P gene mRNAs by expanding the number of Gs of a conserved A(n)G(n) run. Different viruses insert different distributions of guanylates, e.g., Sendai virus inserts a single G, whereas parainfluenza virus type 3 inserts one to six Gs. The sequences conserved at the editing site, as well as the experimental evidence, suggest that the insertions occur by a stuttering process, i.e., by pseudotemplated transcription. The number of times the polymerase “stutters” at the editing site before continuing strictly templated elongation is directed by a cis-acting sequence found upstream of the insertions. We have examined the stuttering process during natural virus infections by constructing recombinant Sendai viruses with mutations in their cis-acting sequences. We found that the template stutter site is precisely determined (C(1052)) and that a relatively short region (∼6 nucleotides) just upstream of the A(n)G(n) run can modulate the overall frequency of mRNA editing as well as the distribution of the nucleotide insertions. The positions more proximal to the 5′ A(n)G(n) run are the most important in this respect. We also provide evidence that the stability of the mRNA/template hybrid plays a determining role in the overall frequency and range of mRNA editing. When the template U run is extended all the way to the stutter site, adenylates rather than guanylates are added at the editing site and their distribution begins to resemble the polyadenylation associated with mRNA 3′ end formation by the viral polymerase. Our data suggest how paramyxovirus mRNA editing and polyadenylation are related mechanistically and how editing sites may have evolved from poly(A)-termination sites or vice versa

Sendai Viruses with Altered P, V, and W Protein Expression

AbstractWild-type Sendai virus expresses three proteins containing the N-terminal half of the P protein open reading frame due to mRNA editing; a full-length P protein (ca. 70% of the total), a V protein with the N-terminal half fused to a Cys-rich Zn2+-binding domain (ca. 25% of the total), and a W protein representing the N-terminal half alone (ca. 5% of the total). To examine the role of these proteins in the virus life cycle, we have prepared recombinant viruses in which the normal V mRNA expresses a W protein (V-stop; 70% P, 30% W), one which cannot edit its P gene mRNA (Δ6A; 100% P), and one which overedits its mRNA like parainfluenza virus type 3 (swap/8; 20–40% P, 30% V, 30% W). All these viruses were readily recovered and grew to similar titers in eggs, and except for the P gene products, cell lines individually infected with these viruses accumulated similar amounts of viral macromolecules. The relative competitive advantage of each virus was determined by multiple cycle coinfections of eggs and found to be rSeV-Vstop= rSeV-wt ⪢ rSeV-Δ6A > rSeV-swap/8. On the other hand, rSeV-swap/8 underwent multiple cycles of replication in C57Bl/6 mouse lungs and was highly virulent for these animals, whereas rSeV-Δ6A was avirulent in mice and this infection was quickly cleared. Remarkably, rSeV-Vstopappeared to be more virulent for inbred C57Bl/6 mice than rSeV-wt, but was partially attenuated in infections of outbred ICR mice. Thus, the expression of either the V or the W proteins is sufficient for multiple cycles of infection and pathogenesis in C57Bl/6 mice, whereas W can only partially substitute for V for pathogenesis in ICR mice

Partial characterization of a Sendai virus replication promoter and the rule of six

We have used a cDNA copy of a natural, internally deleted, Sendai virus defective interfering genome to study the effect of insertions and deletions (which maintain the hexamer genome length) on the ability of viral genomes to be amplified in a transfected cell system. The insertion of 18 nt at nt72 (In the 5' untranslated region of the N gene, just downstream of the le+ region) was found to be lethal, whereas similar insertions further from the genome ends were well tolerated. Curiously, the insertion of 6 nt on either side of the le+/N junction (at nt47 and nt87) was well tolerated, but the insertion of 12 nt at either site, or of 6 nt at both sites, largely ablated genome amplification. These results suggest that an element of this replication promoter is located downstream of nt87, in the 5' untranslated region of the first gene. Remarkably, the addition of 6 nt by the insertion of 2, 3, or 4 nt at nt47 plus the insertion of 4, 3, or 2 nt, respectively, at nt87 was poorly tolerated, presumably because the hexamer phase of the intervening sequence was altered with respect to the N subunits of the template. These results suggest that the rule of six operates, at least in part, at the level of the initiation of antigenome synthesis

Regeneration of a well-differentiated human airway surface epithelium by spheroid and lentivirus vector-transduced airway cells.

BACKGROUND: Following injury to the airway epithelium, rapid regeneration of a functional epithelium is necessary in order to restore the epithelial barrier integrity. In the perspective of airway gene/cell therapy, we analyzed the capacity of human airway epithelial cells cultured as three-dimensional (3-D) spheroid structures to be efficiently transduced on long term by a pseudotyped lentiviral vector. The capacity of the 3-D spheroid structures to repopulate a denuded tracheal basement membrane and regenerate a well-differentiated airway epithelium was also analyzed. METHODS: An HIV-1-derived VSV-G pseudotyped lentiviral vector encoding the enhanced green fluorescent protein (eGFP) was used. Airway epithelial cells were isolated from mature human fetal tracheas and airway xenografts, cultured as 3-D spheroid structures, and either transduced at multiplicity of infection (MOI) 10 and 100 or assayed in an ex vivo and in vivo model to evaluate their regeneration capacity. RESULTS: An in vivo repopulation assay in SCID-hu mice with transduced isolated fetal airway epithelial cells shows that lentiviral transduction does not alter the airway reconstitution. Transduction of the 3-D spheroid structures shows that 12% of cells were eGFP-positive for up to 80 days. In ex vivo and in vivo assays (NUDE-hu mice), the 3-D spheroid structures are able to repopulate denuded basement membrane and reconstitute a well-differentiated human airway surface epithelium. CONCLUSIONS: The efficient and long-term lentiviral transduction of 3-D spheroid structures together with their capacity to regenerate a well-differentiated mucociliary epithelium demonstrate the potential relevance of these 3-D structures in human airway gene/cell therapy