Lentiviral vectors designed for the treatment of the
hemoglobinopathies require the inclusion of regulatory and strong enhancer elements to achieve sufficient expression of the β-
globin transgene. Despite the inclusion of these elements, the efficacy of these vectors may be limited by transgene silencing due to the genomic environment surrounding the integration site. Barrier insulators can be used to give more consistent expression and resist silencing even with lower vector copies. Here, the barrier activity of an insulator element from the human ankyrin-1 gene was analyzed in a lentiviral vector carrying an antisickling human β-
globin gene. Inclusion of a single copy of the
Ankyrin insulator did not affect viral titer, and improved the consistency of expression from the vector in murine
erythroleukemia cells. The presence of the
Ankyrin insulator element did not change transgene expression in human hematopoietic cells in short-term erythroid culture or in vivo in primary murine transplants. However, analysis in secondary recipients showed that the lentiviral vector with the
Ankyrin element preserved transgene expression, whereas expression from the vector lacking the
Ankyrin insulator decreased in secondary recipients. These studies demonstrate that the
Ankyrin insulator may improve long-term β-
globin expression in hematopoietic stem cells for gene therapy of
hemoglobinopathies.