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

Author:

Vacek Marla M.1,Ma Hong1,Gemignani Federica1,Lacerra Giuseppina1,Kafri Tal1,Kole Ryszard1

Affiliation:

1. From the Curriculum in Genetics and Molecular Biology, University of North Carolina Gene Therapy Center, Department of Pharmacology and Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill; International Agency for Research on Cancer, Lyon, France; and Istituto di Genetica e Biofisica Adriano Buzzati Traverso-Consiglio Nazionale delle Ricerche, Naples, Italy.

Abstract

AbstractMutations at nucleotides 654, 705, or 745 in intron 2 of the human β-globin gene activate aberrant 3′ and 5′ splice sites within the intron and prevent correct splicing of β-globin pre-mRNA, resulting in inhibition of β-globin synthesis and in consequence β-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 β-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 β-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 β-thalassemia.

Publisher

American Society of Hematology

Subject

Cell Biology,Hematology,Immunology,Biochemistry

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