Abstract
Hematopoietic stem cell (HSC) gene therapy is being evaluated for hemoglobin disorders including sickle cell disease (SCD). Therapeutic globin vectors have demanding requirements including high-efficiency transduction at the HSC level and high-level, erythroid-specific expression with long-term persistence. The requirement of intron 2 for high-level β-globin expression dictates a reverse-oriented globin-expression cassette to prevent its loss from RNA splicing. Current reverse-oriented globin vectors can drive phenotypic correction, but they are limited by low vector titers and low transduction efficiencies. Here we report a clinically relevant forward-oriented β-globin-expressing vector, which has sixfold higher vector titers and four to tenfold higher transduction efficiency for long-term hematopoietic repopulating cells in humanized mice and rhesus macaques. Insertion of Rev response element (RRE) allows intron 2 to be retained, and β-globin production is observed in transplanted macaques and human SCD CD34+ cells. These findings bring us closer to a widely applicable gene therapy for hemoglobin disorders.
Highlights
Hematopoietic stem cell (HSC) gene therapy is being evaluated for hemoglobin disorders including sickle cell disease (SCD)
Current globin-expressing vectors are all reverse-oriented to prevent the loss of intron 2 by RNA splicing during viral preparation, as intron 2 is required for high-level β-globin expression[15]
We hypothesized that reverse orientation impedes both viral preparation and vector transduction, as despite extensive modifications through deletion of cryptic polyadenylation signals to optimize a conventional reverse-oriented globin vector, ~10-fold lower titers and less efficient transduction in human CD34+ cells were still observed as compared with a standard forward-oriented gene marking vector (Supplementary Note 1 and Supplementary Fig. 1)
Summary
Hematopoietic stem cell (HSC) gene therapy is being evaluated for hemoglobin disorders including sickle cell disease (SCD). Similar in vitro GFP or YFP RNA expression was observed in the forward-oriented and standard marking vectors in differentiated erythroid cells from both animals (Supplementary Fig. 6).
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