Figure 1.

Maps of AnkT9W, AnkT9W-derived ALS16–20 vectors, and clinical vectors (CVs) engineered in other laboratories

Viral features—LTR, long terminal repeat; SD, splice donor; SA, splice acceptor; RRE, reverse responsive element; sinLTR, self-inactivating long terminal repeat. Transgene features—e⁺, β-globin gene enhancer; βp, β-globin gene promoter; HS, hypersensitive site; 3′ UTR, 3′ untranslated region of the β-globin gene. β-globin gene exons and introns are indicated by red and blue segments, respectively. The length of the segments from the β-globin gene promoter to the end of the enhancer and the mini LCR are indicated under each segment. T87Q indicates a threonine to glutamine substitution at amino acid 87. Miscellaneous features—ankyrin, human ankyrin gene insulator element. The region indicated as a Δ+ comprises a 374-bp DNA sequence that was reinstated to reproduce the wild-type intron II (850 bp) in all ALS vectors. Note, WPRE, woodchuck hepatitis post-transcriptional regulatory element; pA, bovine growth hormone polyadenylation sequence, which are not included in the diagrams but are present in these constructs downstream of the sinLTR (see Figure S1 for reference). More specifically, five main modifications of the AnkT9W vector were performed to make ALS17. First, we replaced the truncated intravenous 2 (IVS2) sequence with the full endogenous IVS2 sequence. Second, we removed the woodchuck post-regulatory element (WPRE) from the vector integration sequence and placed it directly after the 3′ LTR. Third, we placed a strong bovine growth hormone poly(A) signal after the WPRE. Fourth, we altered the β-globin gene to express β-globin with the T87Q modification (threonine to glutamine substitution at amino acid 87) instead of endogenous β-globin. Last, we extended the hypersensitive site (HS)4 (1,157 bp) and removed a short polylinker from the 3′ LTR flanking the ankyrin sequence. For ALS16, we replaced the ALS17 LCR with that of the CV-I vector, as previously published. In ALS18, we swapped the 500-bp promoter from ALS16 with a shorter version (~200 bp) to asses if the shorter β-globin promoter was equally efficient in transcribing the β-globin gene. ALS19 and ALS20 have the same elements as ALS16 and also include the core element of HS1 (556 bp), as previously reported²⁵ in either sense or antisense orientation.