Specific DNA sequences cause premature polyadenylation of globin vector genomic RNA and compromise vector titer. (A) Schematic representations of γ-globin lentiviral vectors. Shown at top is the SJ-1 self-inactivating (SIN) lentiviral vector backbone which contains both the central polypurine tract (cppt) and the rev-responsive element (RRE).13 The genomic γ-globin sequences, as previously described,3 are indicated by the hatched design, the β-globin promoter sequences are indicated by the solid horizontal arrow, and the HS elements, with the size and restriction sites that define the fragments from the β-globin LCR, are represented as horizontal open rectangles. Vertical solid arrowheads indicate the sites of mutagenesis that eliminated canonical “AATAAA” polyadenylation signal sequences. (B) Northern blot analysis, using a radiolabeled RRE probe, of RNA from 293T cells transfected with the indicated vectors.Arrows indicate the predicted size of the full-length vector genomic RNAspecies. Shown on the left are the migration positions of a set of RNA markers of the indicated sizes. (C) 3′ RACE analysis to map premature polyadenylation cleavage sites in mLARβΔγV1 vector genomic RNA transcripts. RNA from 293T cells transfected with the mLARβΔγV1 vector was subjected to 3′ RACE analysis as described in “Materials and methods.” The amplified reaction products, derived using either the GSP1 or GSP2 primers, were fractionated using agarose gel electrophoresis and visualized by ethidium bromide staining (right). The products of each reaction were also subsequently cloned and sequenced. At left, the vertical solid arrows indicate the major sites of aberrant cleavage of vector RNA as determined from sequencing data.