rHS432Aγ*-mediated gene transfer and expression in primary, progenitor-derived, human erythroid colonies. CD34-selected bone marrow cells were exposed to rAAV at various multiplicities of infection and cultured, and individual colonies were analyzed for mRNA at 14 to 16 days. The diagram below shows the design of the RT-PCR analysis; the upper band (369 bp), when present, is derived from genomic or vector DNA regardless of the use of reverse transcriptase (+ or −), whereas the lower (254 bp) reverse transcriptase-dependent band (+) is derived from processed globin mRNA. The 5′ primer designed to detect the Aγ* mRNA spans a 6-bp deletion in the 5′ untranslated region (diagram below), whereas the 5′ primer designed to detect the wild-type γ mRNA includes the deleted sequences (not shown). The upper band with the Aγ* primers in several of the figures is derived from rAAV vector particles, episomal DNA, or possibly integrated rAAV genomic DNA; its presence in the absense of Aγ* gene expression (upper panel, 1.4 × 107 MOI) suggests that this signal may be derived from particle DNA or intracellular, single-stranded DNA.
