Genome editing of HBG1 and HBG2 to induce fetal hemoglobin

Induction of fetal hemoglobin (HbF) via clustered regularly interspaced short palindromic repeats/Cas9-mediated disruption of DNA regulatory elements that repress gamma-globin gene (HBG1 and HBG2) expression is a promising therapeutic strategy for sickle cell disease (SCD) and beta-thalassemia, although the optimal technical approaches and limiting toxicities are not yet fully defined. We disrupted an HBG1/HBG2 gene promoter motif that is bound by the transcriptional repressor BCL11A. Electroporation of Cas9 single guide RNA ribonucleoprotein complex into normal and SCD donor CD34+ hematopoietic stem and progenitor cells resulted in high frequencies of on-target mutations and the induction of HbF to potentially therapeutic levels in erythroid progeny generated in vitro and in vivo after transplantation of hematopoietic stem and progenitor cells into nonobese diabetic/severe combined immunodeficiency/Il2rgamma-/-/KitW41/W41 immunodeficient mice. On-target editing did not impair CD34+ cell regeneration or differentiation into erythroid, T, B, or myeloid cell lineages at 16 to 17 weeks after xenotransplantation. No off-target mutations were detected by targeted sequencing of candidate sites identified by circularization for in vitro reporting of cleavage effects by sequencing (CIRCLE-seq), an in vitro genome-scale method for detecting Cas9 activity. Engineered Cas9 containing 3 nuclear localization sequences edited human hematopoietic stem and progenitor cells more efficiently and consistently than conventional Cas9 with 2 nuclear localization sequences. Our studies provide novel and essential preclinical evidence supporting the safety, feasibility, and efficacy of a mechanism-based approach to induce HbF for treating hemoglobinopathies

BCL2A1a Over-Expression in Murine Hematopoietic Stem and Progenitor Cells Decreases Apoptosis and Results in Hematopoietic Transformation

We previously reported the development of a lethal myeloid sarcoma in a non-human primate model utilizing retroviral vectors to genetically modify hematopoietic stem and progenitor cells. This leukemia was characterized by insertion of the vector provirus into the BCL2A1 gene, with resultant BCL2A1 over-expression. There is little information on the role of this anti-apoptotic member of the BCL2 family in hematopoiesis or leukemia induction. Therefore we studied the impact of Bcl2a1a lentiviral over-expression on murine hematopoietic stem and progenitor cells. We demonstrated the anti-apoptotic function of this protein in hematopoietic cells, but did not detect any impact of Bcl2a1a on in vitro cell growth or cell cycle kinetics. In vivo, we showed a higher propensity of HSCs over-expressing Bcl2a1a to engraft and contribute to hematopoiesis. Mice over-expressing Bcl2a1a in the hematologic compartment eventually developed an aggressive malignant disease characterized as a leukemia/lymphoma of B-cell origin. Secondary transplants carried out to investigate the primitive origin of the disease revealed the leukemia was transplantable. Thus, Bcl2a1 should be considered as a protooncogene with a potential role in both lymphoid and myeloid leukemogenesis, and a concerning site for insertional activation by integrating retroviral vectors utilized in hematopoietic stem cell gene therapy.intramural research programs of the National Heart Lung and Blood Institute (CED) of the National Institutes of Healthintramural research programs of the National Heart Lung and Blood Institute (CED) of the National Institutes of Healt

Identification de nouveaux partenaires pour les protéïnes LAP humaines Erbin et hScrib

CHEZ L ADULTE, 85 % DES CAS DE CANCERS SONT DES CARCINOMES, QUI ONT DONC POUR ORIGINE DES DEREGULATIONS DE L HOMEOSTASIE EPITHELIALE. TROIS TYPES DE GENES SONT A L ORIGINE DE LA CARCINOGENESE, LES GENES DE LA STABILITE DU GENOME, LES ONCOGENES, ET LES SUPPRESSEURS DE TUMEURS. PARMI CES DERNIERS, LA FAMILLE DES PROTEINES LAP, CONSERVEE AU COURS DE L EVOLUTION, EST IMPLIQUEE DANS LES PROCESSUS DE MISE EN PLACE ET DE MAINTIEN DE LA POLARITE DES CELLULES EPITHELIALES. SON NOM LUI VIENT DES DEUX DOMAINES PROTEIQUES QUI COMPOSENT TOUS SES MEMBRES : UN DOMAINE LRR ET UN A PLUSIEURS DOMAINES PDZ. AU LABORATOIRE NOUS CHERCHONS LE ROLE EXACT QUE JOUENT CES PROTEINES CHEZ L HOMME. UNE DES CARACTERISTIQUES COMMUNES DES PROTEINES LAP EST LEUR LOCALISATION SUBCELLULAIRE MEDIEE PAR LEUR DOMAINE LRR. NOUS AVONS AINSI CHERCHE A DECRYPTER LE MECANISME MOLECULAIRE PAR LEQUEL CES PROTEINES, ET NOTAMMENT ERBIN ET HSCRIB, ETAIENT LOCALISEES A LA MEMBRANE BASOLATERALE DES CELLULES EPITHELIALES. DE NOMBREUSES TECHNIQUES ONT ETE MISES EN ŒUVRE AFIN D IDENTIFIER LE(S) PARTENAIRE(S) DE LOCALISATION. CEPENDANT, COMME LES LABORATOIRES CONCURRENTS NOUS NE SOMMES PAS ARRIVES A NOS FINS. UNE FOIS LOCALISEES A LA MEMBRANE, LES PROTEINES LAP INTERAGISSENT AVEC DES CONSTITUANTS DES JONCTIONS CELLULAIRES. DANS CE MANUSCRIT NOUS DEMONTRONS L INTERACTION ENTRE ERBIN ET ETA-CATENINE, UN CONSTITUANT MAJEUR DES JONCTIONS ADHERENTES, AINSI QUE L INTERACTION ENTRE HSCRIB ET ZO-2, UNE PROTEINE IMPLIQUEE DANS LES JONCTIONS SERREES. APRES AVOIR CARACTERISE LES MODALITES D INTERACTION ENTRE LES PROTEINES DE CES DEUX COMPLEXES, NOUS AVONS CHERCHE LEUR FONCTION DANS LES CELLULES EPITHELIALES.CARCINOMAS REPRESENT 85% OF THE CANCER CASES IN THE ADULT. HENCE, THEY IMPLY EPITHELIAL HOMEOSTASIS DYSREGULATION DUE TO THREE TYPES OF GENES: CARETAKER GENES, ONCOGENES, AND TUMOR SUPPRESSOR GENES. A NEWLY DESCRIBED PROTEIN FAMILY BELONGS TO THE THIRD TYPE. THE LAP PROTEIN FAMILY (FOR LRR AND PDZ DOMAINS) IS CONSERVED THROUGH EVOLUTION AND IS IMPLIED IN THE ESTABLISHMENT AND THE MAINTENANCE OF EPITHELIAL CELL POLARITY. IN THE LABORATORY WE ARE LOOKING FOR THE ROLE OF THE HUMAN LAP MEMBERS IN EPITHELIAL CELLS. A HALLMARK OF THE FAMILY IS THEIR SUBCELLULAR LOCALIZATION MEDIATED BY THE LRR DOMAIN OF EACH MEMBER. WE TRIED TO DECIPHER THE MOLECULAR MECHANISM ALLOWING THE LOCALIZATION OF ERBIN AND HSCRIB TO THE BASOLATERAL MEMBRANE OF EPITHELIAL CELLS. UNFORTUNATELY, LIKE OTHERS, WE USED NUMEROUS TECHNIQUES TO IDENTIFY THE BINDING PARTNER(S) FOR THE LOCALIZATION WITH NO SUCCESS. AFTER REACHING THE BASOLATERAL MEMBRANE, LAP PROTEINS INTERACT WITH COMPONENTS OF THE JUNCTIONNAL COMPLEXES. WE SHOW IN THIS REPORT THAT AN INTERACTION EXISTS BETWEEN ERBIN AND BETA-CATENIN, A MAJOR PROTEIN OF ADHERENS JUNCTIONS AND BETWEEN HSCRIB AND ZO-2, A CYTOSOLIC PROTEIN OF THE TIGHT JUNCTIONS. WE CHARACTERIZED THE MODALITIES OF THESE INTERACTIONS AND LOOKED FOR THEIR FUNCTION IN EPITHELIAL CELLS.AIX-MARSEILLE1-BU Sci.St Charles (130552104) / SudocSudocFranceF

Chapter 16: Mammalian Neogene biostratigraphy of the Sulaiman Province, Pakistan

International audienc

New remains of Lophiaspis maurettei (Mammalia, Perissodactyla) from the early Eocene of France and the implications for the origin of the Lophiodontidae

International audienceThe Lophiodontidae are endemic perissodactyls from Europe that flourished during the Eocene. Despite their preponderance in the European fossil record, their exact origin and relationships within the perissodactyls remain unknown due to the rare and fragmentary material in the early Ypresian, the time of their earliest radiation. Lophiaspis maurettei is the oldest and earliest diverging lophiodontid known to date but is unfortunately poorly known. We describe here the results of new excavations of the type locality of Palette. Important new material including complete skulls, mandibles, post-cranial elements and juvenile specimens lead us to revise Lophiaspis maurettei from Palette and other localities and to describe novel morphology for this species. According to an original phylogenetic analysis, based on a revised matrix of dental, cranio-mandibular and postcranial characters, Ls. maurettei is an early diverging lophiodontid morphologically close to Protomoropus and Paleomoropus, two basal chalicotheres, known from Asia and North America, respectively. Our resulting topology does not support the previously proposed inclusion of the lophiodontids within the Ceratomorpha and supports a position within the suborder Ancylopoda, close to some Eomoropidae representatives. These results imply that Ls. maurettei was restricted to Southern Europe during the early Eocene, which would be compatible with an Asian origin for lophiodontids in accordance with the evolutionary history of other perissodactyls and placental mammal

Integrome signatures of lentiviral gene therapy for SCID-X1 patients.

Lentiviral vector (LV)-based gene therapy holds promise for a broad range of diseases. Analyzing more than 280,000 vector integration sites (VISs) in 273 samples from 10 patients with X-linked severe combined immunodeficiency (SCID-X1), we discovered shared LV integrome signatures in 9 of 10 patients in relation to the genomics, epigenomics, and 3D structure of the human genome. VISs were enriched in the nuclear subcompartment A1 and integrated into super-enhancers close to nuclear pore complexes. These signatures were validated in T cells transduced with an LV encoding a CD19-specific chimeric antigen receptor. Intriguingly, the one patient whose VISs deviated from the identified integrome signatures had a distinct clinical course. Comparison of LV and gamma retrovirus integromes regarding their 3D genome signatures identified differences that might explain the lower risk of insertional mutagenesis in LV-based gene therapy. Our findings suggest that LV integrome signatures, shaped by common features such as genome organization, may affect the efficacy of LV-based cellular therapies

Pre-existing immunity does not impair the engraftment of CRISPR-Cas9-edited cells in rhesus macaques conditioned with busulfan or radiation

CRISPR-Cas9-based therapeutic genome editing approaches hold promise to cure a variety of human diseases. Recent findings demonstrate pre-existing immunity for the commonly used Cas orthologs from Streptococcus pyogenes (SpCas9) and Staphylococcus aureus (SaCas9) in humans, which threatens the success of this powerful tool in clinical use. Thus, a comprehensive investigation and potential risk assessment are required to exploit the full potential of the system. Here, we investigated existence of immunity to SpCas9 and SaCas9 in control rhesus macaques (Macaca mulatta) alongside monkeys transplanted with either lentiviral transduced or CRISPR-SpCas9 ribonucleoprotein (RNP)-edited cells. We observed significant levels of Cas9 antibodies in the peripheral blood of all transplanted and non-transplanted control animals. Transplantation of ex vivo transduced or SpCas9-mediated BCL11A enhancer-edited cells did not alter the levels of Cas9 antibodies in rhesus monkeys. Following stimulation of peripheral blood cells with SpCas9 or SaCas9, neither Cas9-specific T cells nor cytokine induction were detected. Robust and durable editing frequencies and expression of high levels of fetal hemoglobin in BCL11A enhancer-edited rhesus monkeys with no evidence of an immune response (>3 years) provide an optimistic outlook for the use of ex vivo CRISPR-SpCas9 (RNP)-edited cells