Annie Rebibo-Sabbah

Development of novel aminoglycoside (NB54) with reduced toxicity and enhanced suppression of disease-causing premature stop mutations

Nonsense mutations promote premature translational termination and represent the underlying cause of a large number of human genetic diseases. The aminoglycoside antibiotic gentamicin has the ability to allow the mammalian ribosome to read past a false-stop signal and generate full-length functional proteins. However, severe toxic side effects along with the reduced suppression efficiency at subtoxic doses limit the use of gentamicin for suppression therapy. We describe here the first systematic development of the novel aminoglycoside 2 (NB54) exhibiting superior in vitro readthrough efficiency to that of gentamicin in seven different DNA fragments derived from mutant genes carrying nonsense mutations representing the genetic diseases Usher syndrome, cystic fibrosis, Duchenne muscular dystrophy, and Hurler syndrome. Comparative acute lethal toxicity in mice, cell toxicity, and the assessment of hair cell toxicity in cochlear explants further indicated that 2 exhibits far lower toxicity than that of gentamicin.

In vitro and ex vivo suppression by aminoglycosides of PCDH15 nonsense mutations underlying type 1 Usher syndrome

Type 1 Usher syndrome (USH1) is a recessively inherited condition, characterized by profound prelingual deafness, vestibular areflexia, and prepubertal onset of retinitis pigmentosa (RP). While the auditory component of USH1 can be treated by cochlear implants, to date there is no effective treatment for RP. USH1 can be caused by mutations in each of at least six genes. While truncating mutations of these genes cause USH1, some missense mutations of the same genes cause nonsyndromic deafness. These observations suggest that partial or low level activity of the encoded proteins may be sufficient for normal retinal function, although not for normal hearing. In individuals with USH1 due to nonsense mutations, interventions enabling partial translation of a full-length functional protein may delay the onset and/or progression of RP. One such possible therapeutic approach is suppression of nonsense mutations by small molecules such as aminoglycosides. We decided to test this approach as a potential therapy for RP in USH1 patients due to nonsense mutations. We initially focused on nonsense mutations of the PCDH15 gene, underlying USH1F. Here, we show suppression of several PCDH15 nonsense mutations, both in vitro and ex vivo. Suppression was achieved both by commercial aminoglycosides and by NB30, a new aminoglycoside-derivative developed by us. NB30 has reduced cytotoxicity in comparison to commercial aminoglycosides, and thus may be more efficiently used for therapeutic purposes. The research described here has important implications for the development of targeted interventions that are effective for patients with USH1 caused by various nonsense mutations.

Extracellular Vesicles in Hematological Disorders

Extracellular vesicles (EVs), comprised of exosomes, microparticles, apoptotic bodies, and other microvesicles, are shed from a variety of cells upon cell activation or apoptosis. EVs promote clot formation, mediate pro-inflammatory processes, transfer proteins and miRNA to cells, and induce cell signaling that regulates cell differentiation, proliferation, migration, invasion, and apoptosis. This paper will review the contribution of EVs in hematological disorders, including hemoglobinopathies (sickle cell disease, thalassemia), paroxysmal nocturnal hemoglobinuria, and hematological malignancies (lymphomas, myelomas, and acute and chronic leukemias).

PO-46 - Influence of extracellular vesicles derived from AML patients on stem cells and their microenvironment

INTRODUCTION Acute myeloid leukemia (AML) is characterized by rapid growth of leukemic blast cells. Extracellular vesicles (EVs) are shed from normal and pathologic cells and express membrane proteins and antigens, reflecting their cellular origin. AIM To explore whether bone marrow EVs of AML patients originate from blast cells and are capable of influencing hematopoietic stem cells (HSC) in a pseudo-natural microenvironment obtained by co-culture of HSC with mesenchymal stem cells (MSC). MATERIALS AND METHODS Bone marrow (BM) samples were collected from healthy controls and patients with newly diagnosed AML at three time points: diagnosis, nadir and remission. EV concentration, cell origin and expression of coagulation proteins were characterized by FACS. Stem cells were obtained from Ficoll gradient of cord blood (CB) followed by CD34+ isolation. Cord blood stem cells with or without MSC were co-incubated with AML EVs. EV internalization was demonstrated by FACS-AMNIS and confocal microscopy. Mir-125b and -155 expressions in the cells were analyzed by RT-PCR. RESULTS AML patients were enrolled in the study. The total BM-EVs number was higher in patients at first remission compared to controls, while blast EV counts (labeled with anti-CD34, CD33, CD117) were higher in patients at diagnosis compared to controls and to patients in remission. Internalization of CD117+/CD33+ BM-EVs to cord blood stem cells in the presence or absence of MSC was evaluated by FACS-AMNIS. Confocal microscopy of CD33+ stained EVs strengthens the findings and shows presence of EVs even in the cytoplasm and the nucleus. Quantitative analysis of mir-125b and mir-155 expression in cord blood stem cells incubated with AML EVs revealed a clear tendency of increased expression in case of cell exposure to AML EVs in comparison to healthy control EVs. This tendency was emphasized in the presence of MSC. CONCLUSIONS EVs of AML patients are generated from blast cells. By internalization into naïve stem cells they can influence their differentiation. Moreover, the presence of mesenchymal stem cells is likely to be essential to the process.

Extracellular Vesicle Characteristics in β-thalassemia as Potential Biomarkers for Spleen Functional Status and Ineffective Erythropoiesis

β-thalassemia major (β-TM) is a therapeutically challenging chronic disease in which ineffective erythropoiesis is a main pathophysiological factor. Extracellular vesicles (EVs) are membrane-enclosed vesicles released by cells into biological fluids; they are involved in intercellular communication and in multiple physiological and pathological processes. The chaperone heat-shock protein 70 (HSP70), which is released from cells via EVs, aggravates ineffective erythropoiesis in β-TM. We propose that β-TM EVs may show specific signatures, reflecting disease mechanisms, stages and severity. Our study aims were to define EV profiles in β-TM patients, investigate the influence of hypersplenism and splenectomy on EV features, and explore the association of circulating EVs with ineffective erythropoiesis and iron-overload parameters. We characterized circulating EVs in 35 transfusion-dependent β-thalassemia patients and 35 controls using several techniques. Nanoparticle-tracking analysis revealed increased EV concentration in patients vs. controls (P = 0.0036), with smaller EV counts and sizes in patients with hypersplenism. Flow cytometry analysis showed lower levels of RBC and monocyte EVs in patients vs. controls. RBC-EV levels correlated with patient hematocrit, reflecting degree of anemia. The procoagulant potential of the EVs evaluated by flow cytometry revealed lower levels of endothelial protein C receptor-labeled EVs in patients vs. controls, and increased tissue factor-to-tissue factor pathway inhibitor-labeled EV ratio in splenectomized patients, suggesting a hypercoagulable state. Protein content, evaluated in EV pellets, showed increased levels of HSP70 in patients (P = 0.0018), inversely correlated with transfusion requirement and hemoglobin levels, and positively correlated with reticulocyte, erythropoietin and lactate dehydrogenase levels. This first description of EVs in patients with hypersplenism reveals the spleen’s importance in EV physiology and clearance. Circulating EV-HSP70 levels were associated with markers of ineffective erythropoiesis, hemolysis and hematological disease severity. EV analysis in β-TM—reflecting spleen status, hypercoagulability state and ineffective erythropoiesis—may serve as a biomarker of disease dynamics, supporting both anticipation of the risk of complications and optimizing treatment.