High-Level Globin Gene Expression Mediated by a Recombinant Adeno- Associated Virus Genome That Contains the 3* g Globin Gene Regulatory Element and Integrates as Tandem Copies in Erythroid Cells

Recombinant adeno-associated virus (rAAV) vectors are betegrated as a single copy with expression at approximately ing evaluated for gene therapy applications. Using purified 50% the level of an endogenous g globin gene. A second rAAV containing a mutationally marked globin gene (g*) vector, rHS32g*3*RE, containing the regulatory element and sites 2, 3, and 4 from the locus control region (RE) from 3* to the chromosomal g globin gene, integrated (rHS432g*), but lacking a drug-resistance gene, we investias an intact, tandem head to tail concatamer with a median gated the relationship between multiplicity of infection copy number of 6 with variable expression per copy ranging (MOI), gene expression, and unselected genome integration from approximately onefold to threefold that of an endogein erythroid cells. Most primary erythroid progenitors were nous g globin gene. These results establish that purified transduced as reflected by g* mRNA in mature colonies but rAAV can be used to achieve integration and functional exonly at an MOI of greater than 5 Ì 10. Using immortalized pression of a globin gene in erythroid cells, but only when erythroleukemia cells as a model, we found that fewer than high MOIs are used. one half of the colonies that contained the g* transcript q 1997 by The American Society of Hematology. had an integrated, intact rHS432g* genome. rHS432g* in-