High-level globin gene expression mediated by a recombinant adeno-associated virus genome that contains the 3' gamma globin gene regulatory element and integrates as tandem copies in erythroid cells.
Recombinant adeno-associated virus (rAAV) vectors are being evaluated for gene therapy applications. Using purified rAAV containing a mutationally marked globin gene (A(gamma)*) and sites 2, 3, and 4 from the locus control region (rHS432A(gamma)*), but lacking a drug-resistance gene, we investigated the relationship between multiplicity of infection (MOI), gene expression, and unselected genome integration in erythroid cells. Most primary erythroid progenitors were transduced as reflected by A(gamma)* mRNA in mature colonies but only at an MOI of greater than 5 x 10(7). Using immortalized erythroleukemia cells as a model, we found that fewer than one half of the colonies that contained the A(gamma)* transcript had an integrated, intact rHS432A(gamma)* genome. rHS432A(gamma)* integrated as a single copy with expression at approximately 50% the level of an endogenous gamma globin gene. A second vector, rHS32A(gamma)*3'RE, containing the regulatory element (RE) from 3' to the chromosomal A(gamma) globin gene, integrated as an intact, tandem head to tail concatamer with a median copy number of 6 with variable expression per copy ranging from approximately onefold to threefold that of an endogenous y globin gene. These results establish that purified rAAV can be used to achieve integration and functional expression of a globin gene in erythroid cells, but only when high MOIs are used.
Hargrove
P W
PW
Department of Hematology/Oncology, St Jude Children's Research Hospital, Memphis, TN 38105, USA.
Vanin
E F
EF
Kurtzman
G J
GJ
Nienhuis
A W
AW
D000229:Dependovirus / Q000235:genetics*; D004912:Erythrocytes / Q000378:metabolism* / Q000821:virology*; D015672:Erythroid Precursor Cells / Q000378:metabolism / Q000821:virology; D015967:Gene Expression Regulation, Viral; D005822:Genetic Vectors; D016679:Genome, Viral; D005914:Globins / Q000235:genetics*; D006801:Humans; D004915:Leukemia, Erythroblastic, Acute; D012045:Regulatory Sequences, Nucleic Acid; D012091:Repetitive Sequences, Nucleic Acid; D014407:Tumor Cells, Cultured; D016662:Virus Integration / Q000235:genetics
D005914:Globins