Identification of a Novel Core Promoter Element That Enhances Transcription: Application to Ankyrin Promoters for Globin Gene Therapy.
Authors: Laflamme Karina , Elnitski Laura , Owen Ashley N. , Gallagher Patrick G. , Bodine David M. 
Published: October 2019

Abstract
Gene Therapy for the hemoglobin β-chain disorders, Sickle Cell Disease (SCD) or beta thalassemia (β-thal), requires both efficient globin gene transfer into hematopoietic stem cells (HSC) as well as expression of globin mRNA and protein at levels that are >20% of the level of α-globin. Expression of the β-like globin genes in either transgenic mice or from RNA virus vectors integrated into the genome of erythroid cells requires sequences from the Locus Control Region (LCR). The LCR sequences contain cryptic splicing and polyadenylation sequences that contribute to inefficient production and low titers of recombinant virus particles. In addition, the powerful LCR enhancer elements pose a significant risk of insertional activation of leukemia genes. We hypothesized that enhancer-independent promoters from other genes expressed in erythrocytes could be used to express sufficient amounts of β-like globin to treat SCD or β-thal. We have focused on the erythroid ankyrin (ANK-1E) promoter, a compact GC-rich promoter with no conserved sequences that is one of the 4 different tissue-specific promoters used to express the ANK-1 gene. We have previously demonstrated that a “double copy” Moloney Leukemia Virus (MLV) vector in which the ANK-1E promoter linked to a γ-globin gene replaced the promoter and enhancer sequences in the MLV Long Terminal Repeat was produced at high titer. In mice repopulated with HSC transduced with the ANK-1E/γ-globin double copy vector, γ-globin mRNA and protein was expressed at uniform level of 7.5 % of α-globin per vector copy (Sabatino et al. PNAS 97:13294–9, 2000). To obtain the 3–4 fold increase in γ-globin expression needed to reach therapeutic levels we have taken advantage of our recent demonstration that patients with a deletion of a TG (−73/−72) dinucleotide in the ANK-1E promoter are ankyrin deficient due to reduced binding of the transcription initiation complex, TFIID (Gallagher et al. Hum Mol Gen 14:2501–9, 2005). We hypothesized that altering the sequence in the TFIID binding region of the ANK-1E promoter would increase TFIID binding, resulting in the increased level of ANK-1E/γ-globin transcription needed for an effective therapy for SCD and β-thal. We generated a library of ANK-1E promoters with degenerate sequence in the TFIID binding region, preserving the critical TG dinucleotide (NNNNNTGNN). This library of promoters was transcribed in nuclear extract from erythroid K562 cells. The RNA transcripts were cloned by 5′RACE and analyzed by sequencing. Four different sequences were obtained from the sequencing results: the wild type sequence (TGCGGTGAG), GGCGGTGAG, GCCGGTGAG and GGGGGTGAG. The consensus sequence derived from these clones: (T/G)(G/C)(G/C)GG(T/A)GAG was found in similar locations relative to the transcriptional start site in the three other ANK-1 promoter regions, as well as in 22% of >4000 human loci with GC-rich promoters that lack TATA boxes and other consensus promoter sequences. ANK-1E promoters containing the novel sequences were linked to a luciferase reporter gene and tested individually in transient and stable transfection assays in K562 cells. The GCCGGTGAG and GGCGGTGAG promoters expressed 7- and 2.5-fold higher levels of the luciferase mRNA and protein than the wild type promoter (p=0.001; 0.005 respectively). We are evaluating the ability of these promoters to direct higher levels of γ-globin expression in primary mouse erythroid cells.