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Epidermolysis bullosa simplex (EBS), an autosomal dominant skin disorder, is characterized by skin fragility. Genetically, the majority of cases are related to missense sequence variations in two keratin genes K5 or K14, leading to cytolysis of basal keratinocytes (KCs) and intraepidermal blistering. Progress toward the identification of treatments has been hampered by an incomplete understanding of the mechanisms underlying this disease and availability of relevant and reliable in vitro models recapitulating the physiopathological mechanisms. Recent advances in stem cell field have fueled the prospect that these limitations could be overcome, thanks to the availability of disease-specific human induced pluripotent stem cells (hiPSCs). In this study, we generated hiPSC-derived KCs from patients carrying keratin gene K5-dominant sequence variations and compared them with nonaffected hiPSC-derived KCs as well as their primary counterparts. Our results showed that EBS hiPSC-derived KCs displayed proliferative defects, increased capacity to migrate, alteration of extracellular signal‒regulated kinase signaling pathway, and cytoplasmic keratin filament aggregates as observed in primary EBS KCs. Of interest, EBS hiPSC-derived KCs exhibited downregulation of hemidesmosomal proteins, revealing the different effects of keratin gene K5 sequence variations on keratin cytoskeletal organization. With a combination of culture miniaturization and treatment with the chaperone molecule 4-phenylbutyric acid, our results showed that hiPSC-derived KCs represent a suitable model for identifying novel therapies for EBS.

International audience; Gene delivery into hCD34+ hematopoietic stem/progenitor cells (HSPCs) using human immunodeficiency virus, type 1-derived lentiviral vectors (LVs) has several promising therapeutic applications. Numerous clinical trials are currently underway. However, the efficiency, safety, and cost of LV gene therapy could be ameliorated by enhancing target cell transduction levels and reducing the amount of LV used on the cells. Several transduction enhancers already exist, such as fibronectin fragments or cationic compounds. Recently, we discovered Vectofusin-1, a new transduction enhancer, also called LAH4-A4, a short histidine-rich amphipathic peptide derived from the LAH4 family of DNA transfection agents. Vectofusin-1 enhances the infectivity of lentiviral and gamma-retroviral vectors pseudotyped with various envelope glycoproteins. In this study, we compared a family of Vectofusin-1 isomers and showed that Vectofusin-1 remains the lead peptide for HSPC transduction enhancement with LVs pseudotyped with vesicular stomatitis virus glycoproteins and also with modified gibbon ape leukemia virus glycoproteins. By comparing the capacity of numerous Vectofusin-1 variants to promote the modified gibbon ape leukemia virus glycoprotein-pseudotyped lentiviral vector infectivity of HSPCs, the lysine residues on the N-terminal extremity of Vectofusin-1, a hydrophilic angle of 140 degrees formed by the histidine residues in the Schiffer-Edmundson helical wheel representation, hydrophobic residues consisting of leucine were all found to be essential and helped to define a minimal active sequence. The data also show that the critical determinants necessary for lentiviral transduction enhancement are partially different from those necessary for efficient antibiotic or DNA transfection activity of LAH4 derivatives. In conclusion, these results help to decipher the action mechanism of Vectofusin-1 in the context of hCD34+ cell-based gene therapy.

International audience; Autophagy-related proteins such as Beclin-1 are involved in an array of complex processes, including antiviral responses, and may also modulate the efficiency of gene therapy viral vectors. The Tat-Beclin-1 (TB1) peptide has been reported as an autophagy-inducing factor inhibiting the replication of pathogens such as HIV, type 1 (HIV-1). However, autophagy-related proteins are also essential for the early steps of HIV-1 infection. Therefore, we examined the effects of the Beclin-1 evolution-arily conserved domain in TB1 on viral transduction and autophagy in single-round HIV infection or with nonreplicative HIV-1-derived lentiviral vectors. TB1 enhanced transduction with various pseudotypes but without inducing the autophagy process. TB1 augmented the transduction of human CD34؉ hematopoietic stem/progenitor cells while maintaining their capacity to engraft in vivo into humanized mice. TB1 was as effective as other transduction additives and functioned by enhancing the adhesion and fusion of viral particles with target cells but not their aggregation. We also found that the N-terminal L1 loop was critical for TB1 transduction-enhancing activity. Interestingly, the Tat-Beclin-2 (TB2) peptide, derived from the human Beclin-2 protein, was even more potent than TB1 in promoting viral transduction and infection. Taken together, our findings suggest that the TB1 and TB2 peptides enhance the viral entry step. Tat-Beclin peptides therefore represent a new family of viral transduction enhancers for potential use in gene therapy.

International audience; Gene transfer using lentiviral vectors has therapeutic applications spanning from monogenic and infectious diseases to cancer. Such gene therapy has to be improved by enhancing the levels of viral infection of target cells and/or reducing the amount of lentivirus for greater safety and reduced costs. Vectofusin-1, a recently developed cationic amphipathic peptide with a pronounced capacity to enhance such viral transduction, strongly promotes the entry of several retroviral pseudotypes into target cells when added to the culture medium. To clarify the molecular basis of its action the peptide was investigated on a molecular and a supramolecular level by a variety of biophysical approaches. We show that in culture medium vectofusin-1 rapidly forms complexes in the 10 nm range that further assemble into annular and extended nanofibrils. These associate with viral particles allowing them to be easily pelleted for optimal virus-cell interaction. Thioflavin T fluorescence, circular dichroism and infrared spectroscopies indicate that these fibrils have a unique α-helical structure whereas most other viral transduction enhancers form β-amyloid fibrils. A vectofusin-1 derivative (LAH2-A4) is inefficient in biological assays and does not form nanofibrils, suggesting that supramolecular assembly is essential for transduction enhancement. Our observations define vectofusin-1 as a member of a new class of α-helical enhancers of lentiviral infection. Its fibril formation is reversible which bears considerable advantages in handling the peptide in conditions well-adapted to Good Manufacturing Practices and scalable gene therapy protocols.

International audience; The use of lentiviral vectors (LVs) for gene transfer in research, technological, or clinical applications requires the production of large amounts of vector. Mass production of clinical-grade LVs remains a challenge and limits certain perspectives for therapeutic use. Some improvements in LV production protocols have been possible by acting on multiple steps of the production process. The addition of animal-derived cholesterol to the culture medium of producer cells is known to increase the infectivity of LVs. To avoid the use of this animal-derived product in clinical settings, an alternative approach is to increase de novo the production of cholesterol by overexpressing a crucial cholesterogenic enzyme, namely, 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGCR). This project evaluates the impact of such an approach on the production, infectivity, and stability of LVs. We demonstrated that the overexpression of human HMGCR isoform 1 (hHMGCR1) in LV producer cells efficiently increased de novo cholesterol biosynthesis and enhanced by 2- to 3-fold the physical and infectious titers of LVs. We also observed that LVs produced in hHMGCR1-overexpressing cells were comparable in stability to LVs produced under classical conditions and were capable of transducing human CD34+ hematopoietic stem/progenitor cells efficiently. Interestingly, we also showed that LV production in the absence of fetal calf serum (FCS) but under hHMGCR1-overexpressing conditions allowed a viral production yield comparable to that achieved under classical conditions in high FCS content, leading the way to the establishment of new LV production protocols on adherent cells without serum.

Gene transfer vectors based on retroviridae are increasingly becoming a tool of choice for biomedical research and for the development of biotherapies in rare diseases or cancers. To meet the challenges of preclinical and clinical production, different steps of the production process of self-inactivating γ-retroviral (RVs) and lentiviral vectors (LVs) have been improved (e.g., transfection, media optimization, cell culture conditions). However, the increasing need for mass production of such vectors is still a challenge and could hamper their availability for therapeutic use. Recently, we observed that the use of a neutral pH during vector production is not optimal. The use of mildly acidic pH conditions (pH 6) can increase by two- to threefold the production of RVs and LVs pseudotyped with the vesicular stomatitis virus G (VSV-G) or gibbon ape leukemia virus (GALV) glycoproteins. Here, we describe the production protocol in mildly acidic pH conditions of GALVTR- and VSV-G-pseudotyped LVs using the transient transfection of HEK293T cells and the production protocol of GALV-pseudotyped RVs produced from a murine producer cell line. These protocols should help to achieve higher titers of vectors, thereby facilitating experimental research and therapeutic applications.

International audience; Abstract Human immunodeficiency virus type 1-derived lentiviral vectors (LVs) are becoming major tools for gene transfer approaches. Several gene therapy clinical studies involving LVs are currently ongoing. Industrial production of clinical-grade LVs is therefore an important challenge. Some improvements in LV production protocols have already been possible by acting on multiple steps of the production process like transfection, cell culture, or media optimizations. Yet, the effects of physicochemical parameters such as pH remain poorly studied. Mammalian cell cultures are generally performed at neutral pH, which may not be the optimal condition to produce high quantities of LVs with optimal infectious properties. In this study, we showed that lentiviral transient production in HEK293T cells is inversely dependent on the pH value of the harvesting medium. Infectious and physical titers of LVs pseudotyped with GALVTR or VSV-G glycoproteins are enhanced by two- to threefold at pH 6 compared with neutral conditions. pH 6-produced LVs are highly infectious on cell lines but also on relevant primary target cells like hCD34+ hematopoietic stem/progenitor cells. GALVTR-LV particles produced at pH 6 are highly stable at 37°C and resistant to multiple freeze-thaw cycles. Higher levels of expression of intracellular pr55gag polyproteins are observed within HEK293T producer cells cultured at pH 6. The positive effect of pH 6 conditions is also observed for moloney-derived retroviral vectors produced from NIH-3T3 fibroblasts, arguing that the mildly acidic pH effect is not limited to the lentivirus genus and is reproducible in various producer cell lines. This observation may help us in the design of more effective LV production protocols for clinical applications.

Gamma-glutamyl transpeptidase (GGT) mRNA-IV and polyomavirus enhancer activator 3 (PEA3) mRNA are highly expressed in the initial segment of the rat epididymis, and both are regulated by testicular factors. PEA3 protein in rat initial segment nuclear extracts has been shown to bind to a PEA3/Ets binding motif, which is derived from the partially characterized GGT mRNA-IV promoter region. This suggests that PEA3 may be involved in regulating transcription from the rat GGT mRNA-IV gene promoter in the initial segment. Using DNA oligonucleotide primers and DNA sequencing analysis, an approximately 1500-basepair (bp) DNA sequence at the 5' region of the promoter was obtained. Using transient transfection, PEA3 activated transcription of the rat GGT mRNA-IV promoter only in cultured epididymal cells from the rat initial segment, but not in Cos-1 or NRK-52E cells. Promoter deletion analysis indicated that a PEA3/Ets binding motif between nucleotides -22 and -17 is the functional site for PEA3 to activate transcription of GGT promoter IV and that an adjacent Sp1 binding motif is also required to maintain promoter IV activity in epididymal cells. Transcriptional activation of promoter IV was shown to be epididymal cell-specific and PEA3-specific. In addition, PEA3 may act as a weak repressor for transcription of promoter IV, probably using a PEA3/Ets binding motif(s) distal to the transcription start site. A model of how PEA3 is involved in the regulation of transcription of GGT promoter IV in epididymal cells is proposed.

Gene transfer into hCD34+ hematopoietic stem/progenitor cells (HSPCs) using HIV-1-based lentiviral vectors (LVs) has several therapeutic applications ranging from monogenic diseases, infectious diseases and cancer. In such therapeutic context, the gene therapy could be improved by enhancing transduction levels of target cells and by reducing the amount of LVs used on the cells for greater safety and reduced costs. We recently identified a new cationic amphipathic peptide, Vectofusin-1, with viral transduction enhancing capacity, enabling higher transduction levels with low amounts of LV. Vectofusin-1 promotes the entry of several retroviral pseudotypes into target cells when added to the culture medium and is not toxic to HSPCs. Here, we present the first insights into the mechanism of action of this new transduction enhancer. First, a viral pull down assay showed that viral particles were easily pelleted by low speed centrifugation in presence of Vectofusin-1, suggesting that this latter may form unsoluble nanofibrils, trapping lentiviral particles. Atomic force (AFM) and electron microscopy (EM) of Vectofusin-1 confirmed that this peptide is rapidly forming annular aggregates and nanofibres in culture medium. Furthermore, these fibres were shown to be auto-fluorescent in medium with high-protein content (X-Vivo20), allowing the observation of a nanofibrillar network of Vectofusin-1 using confocal microscopy. Next, Vectofusin-1 was shown to strongly interact with Congo Red, especially in presence of lentiviral particles, but labeling with Thioflavin T was inefficient, suggesting that Vectofusin-1 fibres are not amyloid-type fibrils. Structural studies by circular dichroism confirmed this result. The capacity to form nanofibrils appears to be essential for the mechanism of action of Vectofusin-1 since a defective mutant called LAH2-A4, unable to promote lentiviral transduction (Majdoul S. et al (2016) J. Biol. Chem.), was also unable to form nanofibrils. In conclusion, biophysical, nanoscopic and microscopic observations have helped us to define Vectofusin-1 as a new nanofibrillar peptide capable of enhancing lentiviral transduction of target cells in conditions well-adapted to cGMP and scalable gene therapy protocols.

Top of pageAbstract Retroviral vectors integrate into the genome, providing an efficient, stable and durable transgene expression in diverse cellular types. However, integration can result in cis-acting molecular interactions that deregulate the expression of either the transgene or neighboring host cell genes. In particular, integration of some MLV derived vectors close to protooncogenes has been shown to induce tumorigenesis in mouse and, more worryingly, in humans. The analysis of retroviral integration patterns has recently been approached by several groups who have shown that integration is not random. Both retroviral and lentiviral vectors integrate preferentially into transcribed regions, RV close to the transcription start and LV along the transcription unit. The transcriptional activity of targeted cells can also influence the integration pattern. Previous knowledge of integration site profiles of gene therapy vectors under the various metabolic conditions of the target cells seems to be an essential criterion in the choice of the vectors and the conditions of gene transfer. In addition to integration sites-transcription units correlation, other less obvious biases with other genomic features may exist and could be discovered with a larger integrome analysis. We have developed a new high-throughput method for the rapid analysis of high numbers of lentiviral integration sites called Vector Integration Tag Analysis (VITA). VITA is based on the identification of Genomic Tags associated to the insertion sites which are used as signatures of the integration events. We use the capacity of the tagging MmeI enzyme to cleave DNA at a defined distance of its recognition site, in order to generate 21 and 22-bp long tags. This length is sufficient, in most of cases to identify without ambiguity a unique position in the human genome. Concatenation, cloning and sequencing of tags allow to obtain information about 20|[ndash]|25 insertion sites in a single sequencing reaction. Using VITA, 1349 different insertion sites of a lentiviral vector in HeLa cells were mapped from only 487 sequencing reactions. Accuracy of VITA was tested in a cellular clone with 8 well known lentiviral insertions. Less than 2 % of obtained tags were false positives.

Differentiation of hepatic precursor cells in the biliary lineage has rarely been investigated, owing to the lack of convenient in vitro models. In this study, we used sodium butyrate and culture on Matrigel to promote differentiation of WB-F344 rat liver epithelial cells along the biliary phenotype. This differentiation was assessed by following the expression of phenotypic markers at the protein or mRNA level. Sodium butyrate induced cytokeratin 19 expression and gamma-glutamyltranspeptidase activity, together with a large increase in gamma-glutamyltranspeptidase mRNA IV, a transcript expressed at high levels in biliary cells. We also observed an increase in aquaporin-1 and beta4 integrin mRNAs, encoding two proteins expressed in adult biliary cells. Culture on Matrigel increased cytokeratin 19, gamma-glutamyltranspeptidase, and BDS7 expression in WB-F344 cells which still expressed aquaporin-1 and beta4 integrin. These results show that WB-F344 cells are able to differentiate in vitro along the biliary pathway, making them a candidate model for analyzing the molecular events associated with the hepatoblast-biliary cell transition.

gamma-Glutamyl transpeptidase (GGT), a major enzyme of glutathione (GSH) homeostasis, is often used as a biliary marker to follow the differentiation of hepatic precursor cells. The expression of the GGT gene is driven by different promoters and yields multiple mRNAs, depending on the cell type or the stage of differentiation. In the present study, we analyzed the GGT mRNA expression pattern by quantitative reverse transcriptase-polymerase chain reaction or by in situ hybridization i) in the liver, in vivo, at early stages of development; ii) in oval cells, which proliferate and differentiate into hepatocytes in response to galactosamine injury in vivo; and finally, iii) during hepatoblast differentiation, in vitro. We show that GGT gene transcription originates from promoters P3, P4, and P5 in rat hepatic precursor cells. Differentiation of these cells induces profound alterations in GGT gene expression, leading to extinction of promoters P4 and P5, when they differentiate into the hepatocytic pathway, and to extinction of promoters P3 and P5 when they differentiate into the biliary pathway. This diversity in GGT mRNA expression provides unique molecular probes to follow hepatic precursor cell differentiation. Furthermore, the identification of factors governing GGT P5 and P4 promoter expression should provide further insight into the molecular events that occur as the liver precursor cell differentiates into the hepatic lineages.

Gamma-glutamyl transpeptidase (GGT) is an enzyme located at the external surface of epithelial cells. It initiates extracellular glutathione (GSH) breakdown, provides cells with a local cysteine supply and contributes to maintain intracellular GSH level. GGT expression, highly sensitive to oxidative stress, is a part of the cell antioxidant defense mechanisms. We describe recent advances in GGT gene structure and expression knowledge and put emphasis on the complex transcriptional organization of that gene and its conservation among different species. GGT gene structure has been elucidated in rat and mouse where a single gene is transcribed from multiple promoters into several transcripts which finally yield a unique polypeptidic chain. Analysis of rat, mouse, human and pig cDNA and gene sequences reveals a large conservation of the transcriptional organization of that gene. This complex structure provides flexibility in GGT expression controlled at the promoter level, through multiple regulatory sites, and at RNA level by alternate 5′ untranslated sequences which may create a diversity in the stability and translational efficiency of the different transcripts. In conclusion, transcription of the GGT gene from several promoters offers multiple DNA and RNA targets for various oxidative stimuli and contributes to a broad antioxidant cell defense through GGT induction and subsequent cysteine supply from extracellular glutathione.

Gamma-glutamyl transpeptidase (GGT) activity is commonly used to follow the differentiation of liver precursor cells into the biliary lineage. However, the GGT expression in immature hepatocytes or its induction in adult hepatocytes following diverse carcinogenic or noncarcinogenic treatments has questioned the reliability of GGT expression as a biliary marker. In the present study, we investigated the GGT gene expression from its five different promoters in the late fetal, neonatal, and adult rat liver by Northern blot, reverse transcription-polymerase chain reaction, and in situ hybridization analysis. We show that the GGT activity in the 18-day-old fetus results from the transcription of the gene from the promoter P3 in the hepatocytes. In contrast, the GGT promoter P4 activity appears to be specific of biliary cells in normal as well in cholestatic liver. Thus, sequences unique to the GGT transcripts initiated on these two alternate promoters provide unique molecular probes to discriminate between the biliary and the hepatocytic phenotypes in liver differentiation and cell lineage studies.

International audience; Group II introns are self-splicing mobile elements found in prokaryotes and eukaryotic organelles. These introns propagate by homing into precise genomic locations, following assembly of a ribonucleoprotein complex containing the intron-encoded protein (IEP) and the spliced intron RNA. Engineered group II introns are now commonly used tools for targeted genomic modifications in prokaryotes but not in eukaryotes. We speculate that the catalytic activation of currently known group II introns is limited in eukaryotic cells. The brown algae Pylaiella littoralis Pl.LSU/2 group II intron is uniquely capable of in vitro ribozyme activity at physiological level of magnesium but this intron remains poorly characterized. We purified and characterized recombinant Pl.LSU/2 IEP. Unlike most IEPs, Pl.LSU/2 IEP displayed a reverse transcriptase activity without intronic RNA. The Pl.LSU/2 intron could be engineered to splice accurately in Saccharomyces cerevisiae and splicing efficiency was increased by the maturase activity of the IEP. However, spliced transcripts were not expressed. Furthermore, intron splicing was not detected in human cells. While further tool development is needed, these data provide the first functional characterization of the PI.LSU/2 IEP and the first evidence that the Pl.LSU/2 group II intron splicing occurs in vivo in eukaryotes in an IEP-dependent manner.

International audience; Recent studies indicate that neurodegenerative processes that appear during childhood and adolescence in individuals with Wolfram syndrome (WS) occur in addition to early brain development alteration, which is clinically silent. Underlying pathological mechanisms are still unknown. We have used induced pluripotent stem cell-derived neural cells from individuals affected by WS in order to reveal their phenotypic and molecular correlates. We have observed that a subpopulation of Wolfram neurons displayed aberrant neurite outgrowth associated with altered expression of axon guidance genes. Selective inhibition of the ATF6α arm of the unfolded protein response prevented the altered phenotype, although acute endoplasmic reticulum stress response—which is activated in late Wolfram degenerative processes—was not detected. Among the drugs currently tried in individuals with WS, valproic acid was the one that prevented the pathological phenotypes. These results suggest that early defects in axon guidance may contribute to the loss of neurons in individuals with WS.