title: The new self-inactivating lentiviral vector for thalassemia gene therapy combining two HPFH activating elements corrects human thalassemic hematopoietic stem cells.
abstract: To address how low titer, variable expression, and gene silencing affect gene therapy vectors for hemoglobinopathies, in a previous study we successfully used the HPFH (hereditary persistence of fetal hemoglobin)-2 enhancer in a series of oncoretroviral vectors. On the basis of these data, we generated a novel insulated self-inactivating (SIN) lentiviral vector, termed GGHI, carrying the (A)γ-globin gene with the -117 HPFH point mutation and the HPFH-2 enhancer and exhibiting a pancellular pattern of (A)γ-globin gene expression in MEL-585 clones. To assess the eventual clinical feasibility of this vector, GGHI was tested on CD34(+) hematopoietic stem cells from nonmobilized peripheral blood or bone marrow from 20 patients with β-thalassemia. Our results show that GGHI increased the production of γ-globin by 32.9% as measured by high-performance liquid chromatography (p=0.001), with a mean vector copy number per cell of 1.1 and a mean transduction efficiency of 40.3%. Transduced populations also exhibited a lower rate of apoptosis and resulted in improvement of erythropoiesis with a higher percentage of orthochromatic erythroblasts. This is the first report of a locus control region (LCR)-free SIN insulated lentiviral vector that can be used to efficiently produce the anticipated therapeutic levels of γ-globin protein in the erythroid progeny of primary human thalassemic hematopoietic stem cells in vitro.
authors.affiliation: Laboratory of Cell and Gene Therapy, Center for Basic Research, Biomedical Research Foundation of the Academy of Athens (BRFAA) , 11527 Athens, Greece.
authors.forename: Eleni
authors.initials: E
authors.lastname: Papanikolaou
authors.forename: Maria
authors.initials: M
authors.lastname: Georgomanoli
authors.forename: Evangelos
authors.initials: E
authors.lastname: Stamateris
authors.forename: Fottes
authors.initials: F
authors.lastname: Panetsos
authors.forename: Markisia
authors.initials: M
authors.lastname: Karagiorga
authors.forename: Panagiotis
authors.initials: P
authors.lastname: Tsaftaridis
authors.forename: Stelios
authors.initials: S
authors.lastname: Graphakos
authors.forename: Nicholas P
authors.initials: NP
authors.lastname: Anagnou
mesh_terms: D018952:Antigens, CD34 / Q000378:metabolism; D017209:Apoptosis; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D004742:Enhancer Elements, Genetic; D041905:Erythroid Cells / Q000378:metabolism; D004920:Erythropoiesis; D005319:Fetal Hemoglobin / Q000235:genetics / Q000378:metabolism*; D018014:Gene Transfer Techniques; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors / Q000008:administration & dosage / Q000235:genetics / Q000378:metabolism; D057809:HEK293 Cells; D006412:Hematopoietic Stem Cells / Q000378:metabolism* / Q000473:pathology; D006801:Humans; D016086:Lentivirus / Q000235:genetics / Q000378:metabolism*; D008562:Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism; D010957:Plasmids / Q000235:genetics / Q000378:metabolism; D017354:Point Mutation; D011401:Promoter Regions, Genetic; D014162:Transfection; D014721:Vesicular stomatitis Indiana virus / Q000235:genetics; D014759:Viral Envelope Proteins / Q000235:genetics / Q000378:metabolism; D038301:Virus Inactivation; D017086:beta-Thalassemia / Q000628:therapy*; D055545:gamma-Globins / Q000235:genetics / Q000378:metabolism
chemical_list: D018952:Antigens, CD34; C028391:G protein, vesicular stomatitis virus; D008562:Membrane Glycoproteins; D014759:Viral Envelope Proteins; D055545:gamma-Globins; D005319:Fetal Hemoglobin