title: Development of lentiviral vectors with optimized transcriptional activity for the gene therapy of patients with Fanconi anemia.
abstract: Fanconi anemia (FA) is an inherited genetic disease characterized mainly by bone marrow failure and cancer predisposition. Although gene therapy may constitute a good therapeutic option for many patients with FA, none of the clinical trials so far developed has improved the clinical status of these patients. We have proposed strategies for the genetic correction of bone marrow grafts from patients with FA, using lentiviral vectors (LVs). Here we investigate the relevance of the expression of FANCA to confer a therapeutic effect in cells from patients with FA-A, the most frequent complementation group in FA. Our data show that relatively weak promoters such as the vav or phosphoglycerate kinase (PGK) promoter confer, per copy of FANCA, physiological levels of FANCA mRNA in lymphoblastoid cell lines, whereas the cytomegalovirus and, more significantly, spleen focus-forming virus (SFFV) promoters mediated the expression of supraphysiological levels of FANCA mRNA. Insertion of the woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) or a mutated WPRE into the 3' region of PGK-FANCA LVs significantly increased FANCA mRNA levels. At the protein level, however, all tested vectors conferred, per copy of FANCA, similar and physiological levels of the protein, except SFFV LVs, which again conferred supraphysiological levels of FANCA. In spite of their different activity, all tested vectors mediated a similar phenotypic correction in FA-A lymphoblastoid cell lines and also in hematopoietic progenitors from patients with FA-A. On the basis of the efficacy and safety properties of PGK LVs, a PGK LV carrying FANCA and a mutant WPRE is proposed as an optimized vector for the gene therapy of patients with FA-A.
authors.affiliation: Division of Hematopoiesis and Gene Therapy, Centro de Investigaciones Energéticas, Medioambientales, y Tecnológicas (CIEMAT), and Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBER-ER), 28040 Madrid, Spain.
authors.forename: Africa
authors.initials: A
authors.lastname: González-Murillo
authors.forename: M Luz
authors.initials: ML
authors.lastname: Lozano
authors.forename: Lara
authors.initials: L
authors.lastname: Alvarez
authors.forename: Ariana
authors.initials: A
authors.lastname: Jacome
authors.forename: Elena
authors.initials: E
authors.lastname: Almarza
authors.forename: Susana
authors.initials: S
authors.lastname: Navarro
authors.forename: José C
authors.initials: JC
authors.lastname: Segovia
authors.forename: Helmut
authors.initials: H
authors.lastname: Hanenberg
authors.forename: Guillermo
authors.initials: G
authors.lastname: Guenechea
authors.forename: Juan A
authors.initials: JA
authors.lastname: Bueren
authors.forename: Paula
authors.initials: P
authors.lastname: Río
mesh_terms: D002460:Cell Line; D005199:Fanconi Anemia / Q000235:genetics / Q000378:metabolism / Q000628:therapy*; D015316:Genetic Therapy; D005822:Genetic Vectors / Q000235:genetics*; D006801:Humans; D009154:Mutation; D011506:Proteins / Q000235:genetics / Q000378:metabolism; D051567:Proto-Oncogene Proteins c-vav; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D013155:Spleen Focus-Forming Viruses / Q000235:genetics / Q000378:metabolism
chemical_list: D011506:Proteins; D051567:Proto-Oncogene Proteins c-vav; D012333:RNA, Messenger; C496618:VAV1 protein, human