Bioactive food components for colorectal cancer prevention and treatment: A good match.

Colorectal cancer (CRC) is the third most frequent cancer worldwide, accounts for about 10% of the total cancer cases, and ranks as the second cause of death by cancer. CRC is more prevalent in developed countries in close causal relation with occidental diets. Due to anatomy, the diet has a strong impact on CRC. High contents in meat are acknowledged risk factors whereas a diet rich in fruits and vegetables is an established CRC protective factor. Fruits and vegetables contain numerous Bioactive Food Components (BFCs), physiologically active food compounds, beneficial on health. Preventive and therapeutic benefits of BFCs in cancer have increasingly been reported over the past 20 years. BFCs show both chemopreventive and anti-tumor properties in CRC but more interestingly, abundant research describes BFCs as enhancers of conventional cancer treatments. Despite these promising results, their clinical transferability is slowed down by bioavailability interrogations and their poorly understood hormetic effect. In this review, we would like to reposition BFCs as well-fitted for applications in CRC. We provide a synthetic overview of trustworthy BFC applications in CRC, with a special highlight on combinatory approaches and conventional cancer treatment potentiation strategies.

Iron chelation rescues hemolytic anemia and skin photosensitivity in congenital erythropoietic porphyria.

Congenital erythropoietic porphyria (CEP) is an inborn error of heme synthesis resulting from uroporphyrinogen III synthase (UROS) deficiency and the accumulation of nonphysiological porphyrin isomer I metabolites. Clinical features are heterogeneous among patients with CEP but usually combine skin photosensitivity and chronic hemolytic anemia, the severity of which is related to porphyrin overload. Therapeutic options include symptomatic strategies only and are unsatisfactory. One promising approach to treating CEP is to reduce the erythroid production of porphyrins through substrate reduction therapy by inhibiting 5-aminolevulinate synthase 2 (ALAS2), the first and rate-limiting enzyme in the heme biosynthetic pathway. We efficiently reduced porphyrin accumulation after RNA interference-mediated downregulation of ALAS2 in human erythroid cellular models of CEP disease. Taking advantage of the physiological iron-dependent posttranscriptional regulation of ALAS2, we evaluated whether iron chelation with deferiprone could decrease ALAS2 expression and subsequent porphyrin production in vitro and in vivo in a CEP murine model. Treatment with deferiprone of UROS-deficient erythroid cell lines and peripheral blood CD34+-derived erythroid cultures from a patient with CEP inhibited iron-dependent protein ALAS2 and iron-responsive element-binding protein 2 expression and reduced porphyrin production. Furthermore, porphyrin accumulation progressively decreased in red blood cells and urine, and skin photosensitivity in CEP mice treated with deferiprone (1 or 3 mg/mL in drinking water) for 26 weeks was reversed. Hemolysis and iron overload improved upon iron chelation with full correction of anemia in CEP mice treated at the highest dose of deferiprone. Our findings highlight, in both mouse and human models, the therapeutic potential of iron restriction to modulate the phenotype in CEP.

Circulating Tumor Cell Clusters: United We Stand Divided We Fall.

The presence of circulating tumor cells (CTCs) and CTC clusters, also known as tumor microemboli, in biological fluids has long been described. Intensive research on single CTCs has made a significant contribution in understanding tumor invasion, metastasis tropism, and intra-tumor heterogeneity. Moreover, their being minimally invasive biomarkers has positioned them for diagnosis, prognosis, and recurrence monitoring tools. Initially, CTC clusters were out of focus, but major recent advances in the knowledge of their biogenesis and dissemination reposition them as critical actors in the pathophysiology of cancer, especially metastasis. Increasing evidence suggests that "united" CTCs, organized in clusters, resist better and carry stronger metastatic capacities than "divided" single CTCs. This review gathers recent insight on CTC cluster origin and dissemination. We will focus on their distinct molecular package necessary to resist multiple cell deaths that all circulating cells normally face. We will describe the molecular basis of their increased metastatic potential as compared to single CTCs. We will consider their clinical relevance as prognostic biomarkers. Finally, we will propose future directions for research and clinical applications in this promising topic in cancer.

Targeted gene therapy in human-induced pluripotent stem cells from a patient with primary hyperoxaluria type 1 using CRISPR/Cas9 technology.

Primary hyperoxaluria type 1 (PH1) is an inherited metabolic disorder caused by a deficiency of the peroxisomal enzyme alanine-glyoxylate aminotransferase (AGT), which leads to overproduction of oxalate by the liver and results in urolithiasis, nephrocalcinosis and renal failure. The only curative treatment for PH1 is combined liver and kidney transplantation, which is limited by the lack of suitable organs, significant complications, and the life-long requirement for immunosuppressive agents to maintain organ tolerance. Hepatocyte-like cells (HLCs) generated from CRISPR/Cas9 genome-edited human-induced pluripotent stem cells would offer an attractive unlimited source of autologous gene-corrected liver cells as an alternative to orthotopic liver transplantation (OLT). Here we report the CRISPR/Cas9 nuclease-mediated gene targeting of a single-copy AGXT therapeutic minigene into the safe harbour AAVS1 locus in PH1-induced pluripotent stem cells (PH1-iPSCs) without off-target inserts. We obtained a robust expression of a codon-optimized AGT in HLCs derived from AAVS1 locus-edited PH1-iPSCs. Our study provides the proof of concept that CRISPR/Cas9-mediated integration of an AGXT minigene into the AAVS1 safe harbour locus in patient-specific iPSCs is an efficient strategy to generate functionally corrected hepatocytes, which in the future may serve as a source for an autologous cell-based gene therapy for the treatment of PH1.

Genetic background influences hepcidin response to iron imbalance in a mouse model of hemolytic anemia (Congenital erythropoietic porphyria).

Clinical severity is heterogeneous among patients suffering from congenital erythropoietic porphyria (CEP) suggesting a modulation of the disease (UROS deficiency) by environmental factors and modifier genes. A KI model of CEP due to a missense mutation of UROS gene present in human has been developed on 3 congenic mouse strains (BALB/c, C57BL/6, and 129/Sv) in order to study the impact of genetic background on disease severity. To detect putative modifiers of disease expression in congenic mice, hematologic data, iron parameters, porphyrin content and tissue samples were collected. Regenerative hemolytic anemia, a consequence of porphyrin excess in RBCs, had various expressions: 129/Sv mice were more hemolytic, BALB/c had more regenerative response to anemia, C57BL/6 were less affected. Iron status and hemolysis level were directly related: C57BL/6 and BALB/c had moderate hemolysis and active erythropoiesis able to reduce iron overload in the liver, while, 129/Sv showed an imbalance between iron release due to hemolysis and erythroid use. The negative control of hepcidin on the ferroportin iron exporter appeared strain specific in the CEP mice models tested. Full repression of hepcidin was observed in BALB/c and 129/Sv mice, favoring parenchymal iron overload in the liver. Unchanged hepcidin levels in C57BL/6 resulted in retention of iron predominantly in reticuloendothelial tissues. These findings open the field for potential therapeutic applications in the human disease, of hepcidin agonists and iron depletion in chronic hemolytic anemia.

Metastasis suppressor NM23 limits oxidative stress in mammals by preventing activation of stress-activated protein kinases/JNKs through its nucleoside diphosphate kinase activity.

NME1 (nonmetastatic expressed 1) gene, which encodes nucleoside diphosphate kinase (NDPK) A [also known as nonmetastatic clone 23 (NM23)-H1 in humans and NM23-M1 in mice], is a suppressor of metastasis, but several lines of evidence-mostly from plants-also implicate it in the regulation of the oxidative stress response. Here, our aim was to investigate the physiologic relevance of NDPK A with respect to the oxidative stress response in mammals and to study its molecular basis. NME1-knockout mice died sooner, suffered greater hepatocyte injury, and had lower superoxide dismutase activity than did wild-type (WT) mice in response to paraquat-induced acute oxidative stress. Deletion of NME1 reduced total NDPK activity and exacerbated activation of the stress-related MAPK, JNK, in the liver in response to paraquat. In a mouse transformed hepatocyte cell line and in primary cultures of normal human keratinocytes, MAPK activation in response to H2O2 and UVB, respectively, was dampened by expression of NM23-M1/NM23-H1, dependent on its NDPK catalytic activity. Furthermore, excess or depletion of NM23-M1/NM23-H1 NDPK activity did not affect the intracellular bulk concentration of nucleoside di- and triphosphates. NME1-deficient mouse embryo fibroblasts grew poorly in culture, were more sensitive to stress than WT fibroblasts, and did not immortalize, which suggested that they senesce earlier than do WT fibroblasts. Collectively, these results indicate that the NDPK activity of NM23-M1/NM23-H1 protects cells from acute oxidative stress by inhibiting activation of JNK in mammal models.-Peuchant, E., Bats, M.-L., Moranvillier, I., Lepoivre, M., Guitton, J., Wendum, D., Lacombe, M.-L., Moreau-Gaudry, F., Boissan, M., Dabernat, S. Metastasis suppressor NM23 limits oxidative stress in mammals by preventing activation of stress-activated protein kinases/JNKs through its nucleoside diphosphate kinase activity.

MYB-GATA1 fusion promotes basophilic leukaemia: involvement of interleukin-33 and nerve growth factor receptors.

Acute basophilic leukaemia (ABL) is a rare subtype of acute myeloblastic leukaemia. We previously described a recurrent t(X;6)(p11;q23) translocation generating an MYB-GATA1 fusion gene in male infants with ABL. To better understand its role, the chimeric MYB-GATA1 transcription factor was expressed in CD34-positive haematopoietic progenitors, which were transplanted into immunodeficient mice. Cells expressing MYB-GATA1 showed increased expression of markers of immaturity (CD34), of granulocytic lineage (CD33 and CD117), and of basophilic differentiation (CD203c and FcϵRI). UT-7 cells also showed basophilic differentiation after MYB-GATA1 transfection. A transcriptomic study identified nine genes deregulated by both MYB-GATA1 and basophilic differentiation. Induction of three of these genes (CCL23, IL1RL1, and NTRK1) was confirmed in MYB-GATA1-expressing CD34-positive cells by reverse transcription quantitative polymerase chain reaction. Interleukin (IL)-33 and nerve growth factor (NGF), the ligands of IL-1 receptor-like 1 (IL1RL1) and neurotrophic receptor tyrosine kinase 1 (NTRK1), respectively, enhanced the basophilic differentiation of MYB-GATA1-expressing UT-7 cells, thus demonstrating the importance of this pathway in the basophilic differentiation of leukaemic cells and CD34-positive primary cells. Finally, gene reporter assays confirmed that MYB and MYB-GATA1 directly activated NTRK1 and IL1RL1 transcription, leading to basophilic skewing of the blasts. MYB-GATA1 is more efficient than MYB, because of better stability. Our results highlight the role of IL-33 and NGF receptors in the basophilic differentiation of normal and leukaemic cells. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Antisense oligonucleotide-based therapy in human erythropoietic protoporphyria.

In 90% of people with erythropoietic protoporphyria (EPP), the disease results from the inheritance of a common hypomorphic FECH allele, encoding ferrochelatase, in trans to a private deleterious FECH mutation. The activity of the resulting FECH enzyme falls below the critical threshold of 35%, leading to the accumulation of free protoporphyrin IX (PPIX) in bone marrow erythroblasts and in red cells. The mechanism of low expression involves a biallelic polymorphism (c.315-48T>C) localized in intron 3. The 315-48C allele increases usage of the 3' cryptic splice site between exons 3 and 4, resulting in the transcription of an unstable mRNA with a premature stop codon, reducing the abundance of wild-type FECH mRNA, and finally reducing FECH activity. Through a candidate-sequence approach and an antisense-oligonucleotide-tiling method, we identified a sequence that, when targeted by an antisense oligonucleotide (ASO-V1), prevented usage of the cryptic splice site. In lymphoblastoid cell lines derived from symptomatic EPP subjects, transfection of ASO-V1 reduced the usage of the cryptic splice site and efficiently redirected the splicing of intron 3 toward the physiological acceptor site, thereby increasing the amount of functional FECH mRNA. Moreover, the administration of ASO-V1 into developing human erythroblasts from an overtly EPP subject markedly increased the production of WT FECH mRNA and reduced the accumulation of PPIX to a level similar to that measured in asymptomatic EPP subjects. Thus, EPP is a paradigmatic Mendelian disease in which the in vivo correction of a common single splicing defect would improve the condition of most affected individuals.

Metabolic correction of congenital erythropoietic porphyria with iPSCs free of reprogramming factors.

Congenital erythropoietic porphyria (CEP) is due to a deficiency in the enzymatic activity of uroporphyrinogen III synthase (UROS); such a deficiency leads to porphyrin accumulation and results in skin lesions and hemolytic anemia. CEP is a candidate for retrolentivirus-mediated gene therapy, but recent reports of insertional leukemogenesis underscore the need for safer methods. The discovery of induced pluripotent stem cells (iPSCs) has opened up new horizons in gene therapy because it might overcome the difficulty of obtaining sufficient amounts of autologous hematopoietic stem cells for transplantation and the risk of genotoxicity. In this study, we isolated keratinocytes from a CEP-affected individual and generated iPSCs with two excisable lentiviral vectors. Gene correction of CEP-derived iPSCs was obtained by lentiviral transduction of a therapeutic vector containing UROS cDNA under the control of an erythroid-specific promoter shielded by insulators. One iPSC clone, free of reprogramming genes, was obtained with a single proviral integration of the therapeutic vector in a genomic safe region. Metabolic correction of erythroblasts derived from iPSC clones was demonstrated by the disappearance of fluorocytes. This study reports the feasibility of porphyria gene therapy with the use of iPSCs.

CRISPR editing to mimic porphyria combined with light: A new preclinical approach for prostate cancer.

Thanks to its very high genome-editing efficiency, CRISPR-Cas9 technology could be a promising anticancer weapon. Clinical trials using CRISPR-Cas9 nuclease to ex vivo edit and alter immune cells are ongoing. However, to date, this strategy still has not been applied in clinical practice to directly target cancer cells. Targeting a canonical metabolic pathway essential to good functioning of cells without potential escape would represent an attractive strategy. We propose to mimic a genetic metabolic disorder in cancer cells to weaken cancer cells, independent of their genomic abnormalities. Mutations affecting the heme biosynthesis pathway are responsible for porphyria, and most of them are characterized by an accumulation of toxic photoreactive porphyrins. This study aimed to mimic porphyria by using CRISPR-Cas9 to inactivate UROS, leading to porphyrin accumulation in a prostate cancer model. Prostate cancer is the leading cancer in men and has a high mortality rate despite therapeutic progress, with a primary tumor accessible to light. By combining light with gene therapy, we obtained high efficiency in vitro and in vivo, with considerable improvement in the survival of mice. Finally, we achieved the preclinical proof-of-principle of performing cancer CRISPR gene therapy.

FGFR3 has tumor suppressor properties in cells with epithelial phenotype.

BACKGROUND: Due to frequent mutations in certain cancers, FGFR3 gene is considered as an oncogene. However, in some normal tissues, FGFR3 can limit cell growth and promote cell differentiation. Thus, FGFR3 action appears paradoxical. RESULTS: FGFR3 expression was forced in pancreatic cell lines. The receptor exerted dual effects: it suppressed tumor growth in pancreatic epithelial-like cells and had oncogenic properties in pancreatic mesenchymal-like cells. Distinct exclusive pathways were activated, STATs in epithelial-like cells and MAP Kinases in mesenchymal-like cells. Both FGFR3 splice variants had similar effects and used the same intracellular signaling. In human pancreatic carcinoma tissues, levels of FGFR3 dropped in tumors. CONCLUSION: In tumors from epithelial origin, FGFR3 signal can limit tumor growth, explaining why the 4p16.3 locus bearing FGFR3 is frequently lost and why activating mutations of FGFR3 in benign or low grade tumors of epithelial origin are associated with good prognosis. The new hypothesis that FGFR3 can harbor both tumor suppressive and oncogenic properties is crucial in the context of targeted therapies involving specific tyrosine kinase inhibitors (TKIs). TKIs against FGFR3 might result in adverse effects if used in the wrong cell context.

Specific High-Sensitivity Enzymatic Reporter UnLOCKing-Mediated Detection of Oncogenic BCR::ABL1 and EGFR Rearrangements.

Advances in molecular medicine have placed nucleic acid detection methods at the center of an increasing number of clinical applications. Polymerase chain reaction (PCR)-based diagnostics have been widely adopted for their versatility, specificity, and sensitivity. However, recently reported clustered regularly interspaced short palindromic repeats-based methods have demonstrated equivalent to superior performance, with increased portability and reduced processing time and cost. In this study, we applied Specific High-Sensitivity Enzymatic Reporter UnLOCKing (SHERLOCK) technology to the detection of oncogenic rearrangements. We implemented SHERLOCK for the detection of BCR::ABL1 mRNA, a hallmark of chronic myeloid leukemia (CML), and EGFR DNA oncogenic alleles, frequently detected in glioblastoma and non-small cell lung cancer (NSCLC). SHERLOCK enabled rapid, sensitive, and variant-specific detection of BCR::ABL1 and EGFR alterations. Compared with the gold-standard PCR-based methods currently used in clinic, SHERLOCK achieved equivalent to greater sensitivity, suggesting it could be a new tool in CML and NSCLC, to detect low level of molecular residual disease.

In vivo gene transfer targeting in pancreatic adenocarcinoma with cell surface antigens.

BACKGROUND: Pancreatic ductal adenocarcinoma is a deadly malignancy resistant to current therapies. It is critical to test new strategies, including tumor-targeted delivery of therapeutic agents. This study tested the possibility to target the transfer of a suicide gene in tumor cells using an oncotropic lentiviral vector. RESULTS: Three cell surface markers were evaluated to target the transduction of cells by lentiviruses pseudotyped with a modified glycoprotein from Sindbis virus. Only Mucin-4 and the Claudin-18 proteins were found efficient for targeted lentivirus transductions in vitro. In subcutaneous xenografts of human pancreatic cancer cells models, Claudin-18 failed to achieve efficient gene transfer but Mucin-4 was found very potent. Human pancreatic tumor cells were modified to express a fluorescent protein detectable in live animals by bioimaging, to perform a direct non invasive and costless follow up of the tumor growth. Targeted gene transfer of a bicistronic transgene bearing a luciferase gene and the herpes simplex virus thymidine kinase gene into orthotopic grafts was carried out with Mucin-4 oncotropic lentiviruses. By contrast to the broad tropism VSV-G carrying lentivirus, this oncotropic lentivirus was found to transduce specifically tumor cells, sparing normal pancreatic cells in vivo. Transduced cells disappeared after ganciclovir treatment while the orthotopic tumor growth was slowed down. CONCLUSION: This work considered for the first time three aspect of pancreatic adenocarcinoma targeted therapy. First, lentiviral transduction of human pancreatic tumor cells was possible when cells were grafted orthotopically. Second, we used a system targeting the tumor cells with cell surface antigens and sparing the normal cells. Finally, the TK/GCV anticancer system showed promising results in vivo. Importantly, the approach presented here appeared to be a safer, much more specific and an as efficient way to perform gene delivery in pancreatic tumors, in comparison with a broad tropism lentivirus. This study will be useful in future designing of targeted therapies for pancreatic cancer.

ON-Target Adverse Events of CRISPR-Cas9 Nuclease: More Chaotic than Expected.

CRISPR-Cas9 is a highly promising technology for clinical development. However, this powerful tool can induce adverse genomic events. The off-target genotoxicity is well described, predictable, detectable, and resolved by the use of new generations of Cas9 nucleases with high fidelity. In contrast, the ON-target genotoxicity due to a DNA double-strand break at the targeted locus is still underestimated. Here, we review several genomic outcomes induced by CRISPR-Cas9 from the insertion/deletion of a few bases to megabase-scale rearrangements. We hope to highlight this barely detectable complex safety concern to promote further studies to understand the mechanisms better, to detect these unwanted events, and to prevent them for the safety management of future CRISPR-Cas9 clinical trials.

An Orthotopic Model of Glioblastoma Is Resistant to Radiodynamic Therapy with 5-AminoLevulinic Acid.

Radiosensitization of glioblastoma is a major ambition to increase the survival of this incurable cancer. The 5-aminolevulinic acid (5-ALA) is metabolized by the heme biosynthesis pathway. 5-ALA overload leads to the accumulation of the intermediate fluorescent metabolite protoporphyrin IX (PpIX) with a radiosensitization potential, never tested in a relevant model of glioblastoma. We used a patient-derived tumor cell line grafted orthotopically to create a brain tumor model. We evaluated tumor growth and tumor burden after different regimens of encephalic multifractionated radiation therapy with or without 5-ALA. A fractionation scheme of 5 × 2 Gy three times a week resulted in intermediate survival [48-62 days] compared to 0 Gy (15-24 days), 3 × 2 Gy (41-47 days) and, 5 × 3 Gy (73-83 days). Survival was correlated to tumor growth. Tumor growth and survival were similar after 5 × 2 Gy irradiations, regardless of 5-ALA treatment (RT group (53-67 days), RT+5-ALA group (40-74 days), HR = 1.57, p = 0.24). Spheroid growth and survival were diminished by radiotherapy in vitro, unchanged by 5-ALA pre-treatment, confirming the in vivo results. The analysis of two additional stem-like patient-derived cell lines confirmed the absence of radiosensitization by 5-ALA. Our study shows for the first time that in a preclinical tumor model relevant to human glioblastoma, treated as in clinical routine, 5-ALA administration, although leading to important accumulation of PpIX, does not potentiate radiotherapy.

Neonatal bone marrow transplantation prevents liver disease in a murine model of erythropoietic protoporphyria.

BACKGROUND & AIMS: Erythropoietic protoporphyria (EPP) is an inherited disorder of heme biosynthesis caused by partial ferrochelatase deficiency, resulting in protoporphyrin IX (PPIX) accumulation in erythrocytes, responsible for skin photosensitivity. In some EPP patients, the development of cholestatic liver injury due to PPIX accumulation can lead to hepatic failure. In adult EPP mice, bone marrow transplantation (BMT) leads to skin photosensitivity correction but fails to reverse liver damages, probably because of the irreversible nature of liver fibrosis. Our aim was to determine the time course of liver disease progression in EPP mice and to evaluate the protective effect of BMT into neonates. METHODS: We studied the development of liver disease from birth in EPP mice, in relation with erythroid and hepatic PPIX accumulation. To prevent the development of liver disease, BMT was performed into newborn mice using a novel busulfan-mediated preconditioning assay. RESULTS: We showed that hepatic PPIX accumulates during the first 2 weeks and correlates with the onset of a progressive liver fibrosis in 12-day-old EPP mice. Transplantation of normal congenic hematopoietic stem cells into EPP neonates led to long-term donor hematopoiesis recovery. A full correction of erythroid PPIX accumulation and skin photosensitivity was obtained. Furthermore, five months after neonatal BMT, liver damage was almost completely prevented. CONCLUSIONS: We demonstrated for the first time that BMT could be successfully used to prevent liver disease in EPP mice and suggested that BMT would be an attractive therapeutic option to prevent severe liver dysfunction in EPP patients.

Effective gene therapy of mice with congenital erythropoietic porphyria is facilitated by a survival advantage of corrected erythroid cells.

Achieving long-term expression of a therapeutic gene in a given hematopoietic lineage remains an important goal of gene therapy. Congenital erythropoietic porphyria (CEP) is a severe autosomal-recessive disorder characterized by a deficiency in uroporphyrinogen III synthase (UROS), the fourth enzyme of the heme biosynthetic pathway. We used a recently obtained murine model to check the feasibility of gene therapy in this disease. Lentivirus-mediated transfer of the human UROS cDNA into hematopoietic stem cells (HSCs) from Uros(mut248) mice resulted in a complete and long-term enzymatic, metabolic, and phenotypic correction of the disease, favored by a survival advantage of corrected red blood cells. These results demonstrate that the cure of this mouse model of CEP at a moderate transduction level supports the proof of concept of a gene therapy in this disease by transplantation of genetically modified hematopoietic stem cells.

Analysis of post-transcriptional regulations by a functional, integrated, and quantitative method.

In the past 10 years, transcriptome and proteome analyses have provided valuable data on global gene expression and cell functional networks. However, when integrated,these analyses revealed partial correlations between mRNA expression levels and protein abundance thus suggesting that post-transcriptional regulations may be in part responsible for this discrepancy. In the present work, we report the development of a functional, integrated, and quantitative method to measure post-transcriptional regulations that we named FunREG. This method enables (i) quantitative measure of post-transcriptional regulations mediated by selected 3-untranslated regions and exogenous small interfering-RNA or micro-RNAs and (ii) comparison of these regulatory processes in physiologically relevant systems (e.g. cancer versus primary untransformed cells). We applied FunREG to the study of liver cancer, and we demonstrate for the first time the differential regulatory mechanisms controlling gene expression at a post-transcriptional level in normal and tumoral hepatic cells. As an example, translation efficiency mediated by heparin-binding epidermal growth factor 3-untranslated region was increased 3-fold in liver cancer cells compared with normal hepatocytes, whereas stability of an mRNA containing a portion of Cyclin D1 3-untranslated region was increased more than 2-fold in HepG2 cells compared with normal hepatocytes. Consequently we believe that the method presented herein may become an important tool in fundamental and medical research. This approach is convenient and easy to perform, accessible to any investigator, and should be adaptable to a large number of cell type, functional and chemical screens, as well as genome scale analyses. Finally FunREG may represent a helpful tool to reconcile transcriptome and proteome data.

Recent Progress in Genome Editing for Gene Therapy Applications: The French Perspective.

Recent advances in genome editing tools, especially novel developments in the clustered regularly interspaced short palindromic repeats associated to Cas9 nucleases (CRISPR/Cas9)-derived editing machinery, have revolutionized not only basic science but, importantly, also the gene therapy field. Their flexibility and ability to introduce precise modifications in the genome to disrupt or correct genes or insert expression cassettes in safe harbors in the genome underline their potential applications as a medicine of the future to cure many genetic diseases. In this review, we give an overview of the recent progress made by French researchers in the field of therapeutic genome editing, while putting their work in the general context of advances made in the field. We focus on recent hematopoietic stem cell gene editing strategies for blood diseases affecting the red blood cells or blood coagulation as well as lysosomal storage diseases. We report on a genome editing-based therapy for muscular dystrophy and the potency of T cell gene editing to increase anticancer activity of chimeric antigen receptor T cells to combat cancer. We will also discuss technical obstacles and side effects such as unwanted editing activity that need to be surmounted on the way toward a clinical implementation of genome editing. We propose here improvements developed today, including by French researchers to overcome the editing-related genotoxicity and improve editing precision by the use of novel recombinant nuclease-based systems such as nickases, base editors, and prime editors. Finally, a solution is proposed to resolve the cellular toxicity induced by the systems employed for gene editing machinery delivery.

Covichem: A biochemical severity risk score of COVID-19 upon hospital admission.

Clinical and laboratory predictors of COVID-19 severity are now well described and combined to propose mortality or severity scores. However, they all necessitate saturable equipment such as scanners, or procedures difficult to implement such as blood gas measures. To provide an easy and fast COVID-19 severity risk score upon hospital admission, and keeping in mind the above limits, we sought for a scoring system needing limited invasive data such as a simple blood test and co-morbidity assessment by anamnesis. A retrospective study of 303 patients (203 from Bordeaux University hospital and an external independent cohort of 100 patients from Paris Pitié-Salpêtrière hospital) collected clinical and biochemical parameters at admission. Using stepwise model selection by Akaike Information Criterion (AIC), we built the severity score Covichem. Among 26 tested variables, 7: obesity, cardiovascular conditions, plasma sodium, albumin, ferritin, LDH and CK were the independent predictors of severity used in Covichem (accuracy 0.87, AUROC 0.91). Accuracy was 0.92 in the external validation cohort (89% sensitivity and 95% specificity). Covichem score could be useful as a rapid, costless and easy to implement severity assessment tool during acute COVID-19 pandemic waves.