HIGH-LEVEL GLOBIN GENE-EXPRESSION MEDIATED BY A RECOMBINANT ADENOASSOCIATED 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 mutati onally marked globin gene ((A) gamma Y) and sites 2, 3, and 4 from th e locus control region (rHS432(A) gamma), but lacking a drug-resistan ce gene, we investigated the relationship between multiplicity of infe ction (MOI), gene expression, and unselected genome integration in ery throid cells, Most primary erythroid progenitors were transduced as re flected by A gamma mRNA in mature colonies but only at an MOI of grea ter than 5 x 10(7), Using immortalized erythroleukemia cells as a mode l, we found that fewer than one half of the colonies that contained th e A gamma transcript had an integrated, intact rHS432(A) gamma* genom e, rHS432(A) gamma integrated as a single copy with expression at app roximately 50% the level of an endogenous gamma globin gene, A second vector, rHS32(A) gamma3'RE, containing the regulatory element (RE) fr om 3' to the chromosomal (A) gamma globin gene, integrated as an intac t, tandem head to tail concatamer with a median copy number of 6 with variable expression per copy ranging from approximately onefold to thr eefold that of an endogenous gamma globin gene. These results establis h that purified rAAV can be used to achieve integration and functional expression of a globin gene in erythroid cells, but only when high MO Is are used. (C) 1997 by The American Society of Hematology.