Développement des CAR-T dans les tumeurs solides.

DEVELOPMENT OF CAR T-CELLS IN SOLID TUMORS: CHALLENGES AND PERSPECTIVES: While Chimeric Antigen Receptor (CAR) T-cells have shown outstanding results in some hematologic malignancies, studies in solid tumors are less encouraging with poor response rates. Several factors can account for this lack of efficiency in solid tumors: heterogeneous expression or absence of specific target antigen (and so higher risk of toxicity), immunosuppressive microenvironment, homing and tumoral trafficking issues or lack of CAR T-cell persistence. Different approaches can be considered to overcome these resistance mechanisms: bispecific CARs, use of logic gates, combination with immune checkpoint inhibitors, engineered CAR T-cells resistant to immunosuppressive molecules, addition of chemokines or enzymes, combination with oncolytic virus, intra-tumoral administration, selection of memory T cell subpopulations and development of armored CAR T-cells secreting cytokines such as IL-12, -15 or -18. Last generation optimized CAR T-cell design should thus improve therapeutic efficiency. CAR-T cells may represent in a near future a therapeutic breakthrough also in solid tumors, especially in cold tumors and/or tumors lacking MHC class I expression. Cet article fait partie du numéro supplément Les cellules CAR-T : une révolution thérapeutique ? réalisé avec le soutien institutionnel des partenaires Gilead : Kite et Celgene.

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.

Meganuclease-driven targeted integration in CHO-K1 cells for the fast generation of HTS-compatible cell-based assays.

The development of cell-based assays for high-throughput screening (HTS) approaches often requires the generation of stable transformant cell lines. However, these cell lines are essentially created by random integration of a gene of interest (GOI) with no control over the level and stability of gene expression. The authors developed a targeted integration system in Chinese hamster ovary (CHO) cells, called the cellular genome positioning system (cGPS), based on the stimulation of homologous gene targeting by meganucleases. Five different GOIs were knocked in at the same locus in cGPS CHO-K1 cells. Further characterization revealed that the cGPS CHO-K1 system is more rapid (2-week protocol), efficient (all selected clones expressed the GOI), reproducible (GOI expression level variation of 12%), and stable over time (no change in GOI expression after 23 weeks of culture) than classical random integration. Moreover, in all cGPS CHO-K1 targeted clones, the recombinant protein was biologically active and its properties similar to the endogenous protein. This fast and robust method opens the door for creating large collections of cell lines of better quality and expressing therapeutically relevant GOIs at physiological levels, thereby enhancing the potential scope of HTS.

Targeted gene modifications in drug discovery and development.

In the present review, we discuss technologies and principles guiding the choice of cell-based assays. We show that a major trend is to expedite cell line development in cellular systems [stem cells, induced pluripotent stem cells (iPS), primary cells ...] that may reflect the physiology and differentiation of cells in living organs. We discuss different expression technologies and propose that targeted gene integration is the best approach, applicable to all types of cells. We propose that targeted gene expression combined with the development of assays that approach the physiology of the human body, by creating better screening tools, may help to reduce failure and attrition rate at late stages of clinical development.

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.

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.

Lentiviral vectors: optimization of packaging, transduction and gene expression.

Gene transfer vectors based on retroviruses including oncogenic retroviruses and lentiviruses provide effective means for the delivery, integration and expression of exogenous genes in mammalian cells. Lentiviral (LV) vectors provide attractive gene delivery vehicles in the context of non-dividing cells. This review summarizes the different optimized LV genetic systems that have been developed to date. In all cases, the production of LV-derived vectors consists of a genetically split gene expression design. The viral elements that are specifically required are (i). the LV packaging helper proteins consisting of at least the gag-pol genes, (ii). the LV transfer vector RNA containing the transgene expression cassette, and (iii). an heterologous glycoprotein. While the genetic requirements and performances of the two former viral elements will be treated herein, the latter element relative to the envelope pseudotyping of LV vectors will not be further described (cf. review by Cosset in this issue).

A deficiency in dolichyl-P-glucose:Glc1Man9GlcNAc2-PP-dolichyl alpha3-glucosyltransferase defines a new subtype of congenital disorders of glycosylation.

The underlying causes of type I congenital disorders of glycosylation (CDG I) have been shown to be mutations in genes encoding proteins involved in the biosynthesis of the dolichyl-linked oligosaccharide (Glc(3)Man(9)GlcNAc(2)-PP-dolichyl) that is required for protein glycosylation. Here we describe a CDG I patient displaying gastrointestinal problems but no central nervous system deficits. Fibroblasts from this patient accumulate mainly Man(9)GlcNAc(2)-PP-dolichyl, but in the presence of castanospermine, an endoplasmic reticulum glucosidase inhibitor Glc(1)Man(9)GlcNAc(2)-PP-dolichyl predominates, suggesting inefficient addition of the second glucose residue onto lipid-linked oligosaccharide. Northern blot analysis revealed the cells from the patient to possess only 10-20% normal amounts of mRNA encoding the enzyme, dolichyl-P-glucose:Glc(1)Man(9)GlcNAc(2)-PP-dolichyl alpha3-glucosyltransferase (hALG8p), which catalyzes this reaction. Sequencing of hALG8 genomic DNA revealed exon 4 to contain a base deletion in one allele and a base insertion in the other. Both mutations give rise to premature stop codons predicted to generate severely truncated proteins, but because the translation inhibitor emetine was shown to stabilize the hALG8 mRNA from the patient to normal levels, it is likely that both transcripts undergo nonsense-mediated mRNA decay. As the cells from the patient were successfully complemented with wild type hALG8 cDNA, we conclude that these mutations are the underlying cause of this new CDG I subtype that we propose be called CDG Ih.

Congenital disorders of glycosylation type Ig is defined by a deficiency in dolichyl-P-mannose:Man7GlcNAc2-PP-dolichyl mannosyltransferase.

Type I congenital disorders of glycosylation (CDG I) are diseases presenting multisystemic lesions including central and peripheral nervous system deficits. The disease is characterized by under-glycosylated serum glycoproteins and is caused by mutations in genes encoding proteins involved in the stepwise assembly of dolichol-oligosaccharide used for protein N-glycosylation. We report that fibroblasts from a type I CDG patient, born of consanguineous parents, are deficient in their capacity to add the eighth mannose residue onto the lipid-linked oligosaccharide precursor. We have characterized cDNA corresponding to the human ortholog of the yeast gene ALG12 that encodes the dolichyl-P-Man:Man(7)GlcNAc(2)-PP-dolichyl alpha6-mannosyltransferase that is thought to accomplish this reaction, and we show that the patient is homozygous for a point mutation (T571G) that causes an amino acid substitution (F142V) in a conserved region of the protein. As the pathological phenotype of the fibroblasts of the patient was largely normalized upon transduction with the wild type gene, we demonstrate that the F142V substitution is the underlying cause of this new CDG, which we suggest be called CDG Ig. Finally, we show that the fibroblasts of the patient are capable of the direct transfer of Man(7)GlcNAc(2) from dolichol onto protein and that this N-linked structure can be glucosylated by UDP-glucose:glycoprotein glucosyltransferase in the endoplasmic reticulum.