title: Recombinant adeno-associated virus (rAAV)-mediated expression of a human gamma-globin gene in human progenitor-derived erythroid cells.
abstract: Effective gene therapy for the severe hemoglobin (Hb) disorders, sickle-cell anemia and thalassemia, will require an efficient method to transfer, integrate, and express a globin gene in primary erythroid cells. To evaluate recombinant adeno-associated virus (rAAV) for this purpose, we constructed a rAAV vector encoding a human gamma-globin gene (pJM24/vHS432A gamma). Its 4725-nucleotide genome consists of two 180-bp AAV inverted terminal repeats flanking the core elements of hypersensitive sites 2, 3, and 4 from the locus control region of the beta-globin gene cluster, linked to a mutationally marked A gamma-globin gene (A gamma) containing native promoter and RNA processing signals. CD34+ human hematopoietic cells were exposed to rAAV particles at a multiplicity of infection of 500-1000 and cultured in semisolid medium containing several cytokines. A reverse transcriptase polymerase chain reaction assay distinguished mRNA signals derived from transduced and endogenous human gamma-globin genes. Twenty to 40% of human erythroid burst-forming unit-derived colonies expressed the rAAV-transduced A gamma-globin gene at levels 4-71% that of the endogenous gamma-globin genes. The HbF content of pooled control colonies was 26%, whereas HbF was 40% of the total in pooled colonies derived from rAAV transduced progenitors. These data establish that rAAV containing elements from the locus control region linked to a gamma-globin gene are capable of transferring and expressing that gene in primary human hematopoietic cells resulting in a substantial increase in HbF content.
authors.affiliation: National Heart, Lung and Blood Institute, Bethesda, MD 20892.
authors.forename: J L
authors.initials: JL
authors.lastname: Miller
authors.forename: R E
authors.initials: RE
authors.lastname: Donahue
authors.forename: S E
authors.initials: SE
authors.lastname: Sellers
authors.forename: R J
authors.initials: RJ
authors.lastname: Samulski
authors.forename: N S
authors.initials: NS
authors.lastname: Young
authors.forename: A W
authors.initials: AW
authors.lastname: Nienhuis
mesh_terms: D015703:Antigens, CD / Q000032:analysis; D018952:Antigens, CD34; D001483:Base Sequence; D002460:Cell Line; D003001:Cloning, Molecular; D017931:DNA Primers; D000229:Dependovirus / Q000235:genetics*; D015870:Gene Expression; D015316:Genetic Therapy / Q000379:methods; D005822:Genetic Vectors; D005914:Globins / Q000096:biosynthesis*; D006412:Hematopoietic Stem Cells / Q000378:metabolism*; D006801:Humans; D007438:Introns; D008969:Molecular Sequence Data; D010957:Plasmids; D016133:Polymerase Chain Reaction; D011995:Recombination, Genetic; D015183:Restriction Mapping; D014161:Transduction, Genetic; D014162:Transfection
chemical_list: D015703:Antigens, CD; D018952:Antigens, CD34; D017931:DNA Primers; D005914:Globins