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 Y*) and sites 2, 3, and 4 from the locus control region (rHS432(A) 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 rHS432(A) gamma* genome, rHS432(A) gamma* integrated as a single copy with expression at approximately 50% the level of an endogenous gamma globin gene, A second vector, rHS32(A) 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 gamma 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. (C) 1997 by The American Society of Hematology.