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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
PW Hargrove, EF Vanin, GJ Kurtzman and AW Nienhuis
Department of Hematology/Oncology, St Jude Children's Research Hospital,
Memphis, TN 38105, USA.
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.
Volume 89,
Issue 6,
pp. 2167-2175,
03/15/1997
Copyright © 1997 by The American Society of Hematology
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