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

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Abstract
Recombinant adeno-associated virus (rAAV) vectors are being evaluated for gene therapy applications. Using purified rAAV containing a mutationally marked globin gene (Aγ*) and sites 2, 3, and 4 from the locus control region (rHS432Aγ*), 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 Aγ* mRNA in mature colonies but only at an MOI of greater than 5 × 107. Using immortalized erythroleukemia cells as a model, we found that fewer than one half of the colonies that contained the Aγ* transcript had an integrated, intact rHS432Aγ* genome. rHS432Aγ* integrated as a single copy with expression at approximately 50% the level of an endogenous γ globin gene. A second vector, rHS32Aγ*3′RE, containing the regulatory element (RE) from 3′ to the chromosomal Aγ 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 γ 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.

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