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.
Vacek
Marla M
MM
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, 27599, USA.
Ma
Hong
H
Gemignani
Federica
F
Lacerra
Giuseppina
G
Kafri
Tal
T
Kole
Ryszard
R
D015672:Erythroid Precursor Cells / Q000378:metabolism*; D015316:Genetic Therapy; D005822:Genetic Vectors; D005914:Globins / Q000235:genetics; D006367:HeLa Cells; D006441:Hemoglobin A / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D016086:Lentivirus / Q000235:genetics; D009154:Mutation; D012326:RNA Splicing / Q000187:drug effects; D016372:RNA, Antisense / Q000008:administration & dosage / Q000494:pharmacology* / Q000627:therapeutic use; D012342:RNA, Small Nuclear / Q000008:administration & dosage / Q000494:pharmacology* / Q000627:therapeutic use; D013789:Thalassemia / Q000097:blood / Q000235:genetics / Q000628:therapy*; D014162:Transfection
D016372:RNA, Antisense; D012342:RNA, Small Nuclear; C405487:U7 small nuclear RNA; D005914:Globins; D006441:Hemoglobin A