High-level expression of hemoglobin A in human thalassemic erythroid progenitor cells following lentiviral vector delivery of an antisense snRNA.

Mutations at nucleotides 654, 705, or 745 in intron 2 of the human beta-globin gene activate aberrant 3' and 5' splice sites within the intron and prevent correct splicing of beta-globin pre-mRNA, resulting in inhibition of beta-globin synthesis and in consequence beta-thalassemia. Transfection of HeLa cells expressing the 3 thalassemic mutants with modified U7 snRNA (U7.623), containing a sequence antisense to a region between the aberrant splice sites, reduced the incorrect splicing of pre-mRNA and led to increased levels of the correctly spliced beta-globin mRNA and protein. A lentiviral vector carrying the U7.623 gene was effective in restoration of correct splicing in the model cell lines for at least 6 months. Importantly, the therapeutic value of this system was demonstrated in hematopoietic stem cells and erythroid progenitor cells from a patient with IVS2-745/IVS2-1 thalassemia. Twelve days after transduction of the patient cells with the U7.623 lentiviral vector, the levels of correctly spliced beta-globin mRNA and hemoglobin A were approximately 25-fold over background. These results should be regarded as a proof of principle for lentiviral vector-based gene therapy for beta-thalassemia.