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Prepublished online as a Blood First Edition Paper on August 15, 2002; DOI 10.1182/blood-2002-06-1869.
Submitted June 24, 2002
Accepted July 30, 2002
High-level expression of hemoglobin A in human thalassemic erythroid progenitor cells following lentiviral vector delivery of an antisense snRNA
Marla M Vacek, Hong Ma, Federica Gemignani, Giuseppina Lacerra, Tal Kafri, and Ryszard Kole*
Curriculum in Genetics and Molecular Biology, University of North Carolina, Chapel Hill, NC, USA
Gene Therapy Center, University of North Carolina, Chapel Hill, NC, USA
International Agency for Research on Cancer, Lyon, France
Istituto di Genetica e Biofisica Adriano Buzzati Traverso-Consiglio Nazionale delle Ricerche, Naples, Italy
Department of Pharmacology and Linberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, NC, USA
* Corresponding author; email: kole{at}med.unc.edu.
Mutations at nucleotides 654, 705 or 745 in intron 2 of the human ß-globin gene activate aberrant 3' and 5' splice sites within the intron and prevent correct splicing of ß-globin pre-mRNA, resulting in inhibition of ß-globin synthesis and in consequence ß-thalassemia. Transfection of HeLa cells expressing the three 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 b-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 six months. Importantly, the therapeutic value of this system was demonstrated in hematopoietic stem cells and/or 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 ß-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 ß-thalassemia.
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