CD63-GPC1-Positive Exosomes Coupled with CA19-9 Offer Good Diagnostic Potential for Resectable Pancreatic Ductal Adenocarcinoma

Tumor-released extracellular vesicles (EVs) contain tumor-specific cargo distinguishing them from healthy EVs, and making them eligible as circulating biomarkers. Glypican 1 (GPC1)-positive exosome relevance as liquid biopsy elements is still debated. We carried out a prospective study to quantify GPC1-positive exosomes in sera from pancreatic ductal adenocarcinoma (PDAC) patients undergoing up-front surgery, as compared to controls including patients without cancer history and patients displaying pancreatic preneoplasic lesions. Sera were enriched in EVs, and exosomes were pulled down with anti-CD63 coupled magnetic beads. GPC1-positive bead percentages determined by flow cytometry were significantly higher in PDAC than in the control group. Diagnosis accuracy reached 78% (sensitivity 64% and specificity 90%), when results from peripheral and portal blood were combined. In association with echo-guided-ultrasound-fine-needle-aspiration (EUS-FNA) negative predictive value was 80% as compared to 33% for EUS-FNA only. This approach is clinically relevant as a companion test to the already available diagnostic tools, since patients with GPC1-positive exosomes in peripheral blood showed decreased tumor free survival

Preventing Pluripotent Cell Teratoma in Regenerative Medicine Applied to Hematology Disorders

Abstract Iatrogenic tumorigenesis is a major limitation for the use of human induced pluripotent stem cells (hiPSCs) in hematology. The teratoma risk comes from the persistence of hiPSCs in differentiated cell populations. Our goal was to evaluate the best system to purge residual hiPSCs before graft without compromising hematopoietic repopulation capability. Teratoma risk after systemic injection of hiPSCs expressing the reporter gene luciferase was assessed for the first time. Teratoma formation in immune-deficient mice was tracked by in vivo bioimaging. We observed that systemic injection of hiPSCs produced multisite teratoma as soon as 5 weeks after injection. To eliminate hiPSCs before grafting, we tested the embryonic-specific expression of suicide genes under the control of the pmiR-302/367 promoter. This promoter was highly active in hiPSCs but not in differentiated cells. The gene/prodrug inducible Caspase-9 (iCaspase-9)/AP20187 was more efficient and rapid than thymidine kinase/ganciclovir, fully specific, and without bystander effect. We observed that iCaspase-9-expressing hiPSCs died in a dose-dependent manner with AP20187, without reaching full eradication in vitro. Unexpectedly, nonspecific toxicity of AP20187 on iCaspase-9-negative hiPSCs and on CD34+ cells was evidenced in vitro. This toxic effect strongly impaired CD34+-derived human hematopoiesis in adoptive transfers. Survivin inhibition is an alternative to the suicide gene approach because hiPSCs fully rely on survivin for survival. Survivin inhibitor YM155 was more efficient than AP20187/iCaspase-9 for killing hiPSCs, without toxicity on CD34+ cells, in vitro and in adoptive transfers. hiPSC purge by survivin inhibitor fully eradicated teratoma formation in immune-deficient mice. This will be useful to improve the safety management for hiPSC-based medicine.</jats:p

Mutation-Specific Guide RNA for Compound Heterozygous Porphyria On-target Scarless Correction by CRISPR/Cas9 in Stem Cells

International audienceCRISPR/Cas9 is a promising technology for gene correction. However, the edition is often biallelic, and uncontrolled small insertions and deletions (indels) concomitant to precise correction are created. Mutation-specific guide RNAs were recently tested to correct dominant inherited diseases, sparing the wild-type allele. We tested an original approach to correct compound heterozygous recessive mutations. We compared editing efficiency and genotoxicity by biallelic guide RNA versus mutant allele-specific guide RNA in iPSCs derived from a congenital erythropoietic porphyria patient carrying compound heterozygous mutations resulting in UROS gene invalidation. We obtained UROS function rescue and metabolic correction with both guides with the potential of use for porphyria clinical intervention. However, unlike the biallelic one, the mutant allele-specific guide was free of on-target collateral damage. We recommend this design to avoid genotoxicity and to obtain on-target scarless gene correction for recessive disease with frequent cases of compound heterozygous mutations

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 Urosmut248 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

Differential Uridyl-diphosphate-Glucuronosyl Transferase 1A enzymatic arsenal explains the specific cytotoxicity of resveratrol towards tumor colorectal cells

Abstract Resveratrol belongs to the Bioactive Food Component (BFC) family. It seems admitted that its cytotoxic action impacts tumor cells and spares healthy cells, but the published proofs remain rare. We hypothesized that cells may differentially metabolize resveratrol and lead to different systemic impacts. For this, resveratrol metabolization was evaluated by ultra-high-performance liquid chromatography (UHPLC) coupled with diode array detection (DAD), and correlated with the expression of Uridyl-diphosphate-Glucuronosyl Transferase 1A (UGT1A) genes. The expression of UGT1A genes in human colorectal tissues was studied with RNAseq databases. Functional validation of UGT1A enzymes implication in resveratrol sensitivity of colorectal cells established by UGT1A expression modulation. As resveratrol impacts the S phase of the cell cycle, nucleotide metabolic balance was assessed. We found that resveratrol was more cytotoxic in cells with downregulation of UGTs, i.e. tumor cells. Conversely, overexpression of the UGT1A10 gene in an initial resveratrol-sensitive tumor cell line restored the metabolization accompanied by cytotoxicity diminution. Resveratrol affected intestinal sensitive tumor cell homeostasis with a cell growth/proliferation decoupling, cell-cycle modulation, and UXP/AXP nucleotide imbalance resulting in a global reduction of transcription and translation. This impact on global cell activity was restricted to tumor cells. This study improves resveratrol’s general knowledge and explains how its antitumor action can spare non-tumor cells. It also paves the way to select colorectal tumors eligible for resveratrol treatment potentiation without additional toxicity to healthy digestive tissues.</jats:p