Thalassemic splice repair, delivered

The thalassemias constitute one of the most common monogenic disorders and represent a heterogeneous group of anemias characterized by an absence or reduction of one or more of the globin-molecule subunits. The disorders result from a variety of mutations that affect every step of globin biosynthesis. Vacek and colleagues (page 104) describe a novel and innovative technique employing modified U7 small nuclear RNAs (U7 snRNAs) to correct aberrant splicing responsible for a sizable fraction of human thalassemia. Whereas considerable effort has been placed on gene replacement strategies, their method aims to repair, rather than replace, the abnormality. The group has previously shown that antisense constructs targeting aberrant splice sites in the form of oligonucleotides, morpholinos, or U7 snRNAs lead to increased levels of correctly spliced β-globin mRNA by effectively blocking the aberrant splice site, yet delivery of these antisense constructs remained a formidable obstacle to clinical application. (Sierakowska et al, Proc Nat Acad Sci U S A. 1996;93:12840-12844; Gorman et al, Proc Nat Acad Sci U S A. 1998;95:4929-4934; Lacerra et al, Proc Nat Acad Sci U S A. 2000;97;9591-9596). In order to circumvent the need for repeated administration, the group has now constructed lentiviral vectors to permanently deliver the modified U7 snRNAs and has tested the delivery system in both cell lines and primary human cells carrying a thalassemic intron 2 mutation. Two additional cell lines in which GFP expression was interrupted by the mutated globin intron were also employed, an ingenious system allowing monitoring of corrected splicing by fluorescence detection. The lentiviral vector system resulted in correction of abnormal splicing that ranged from 3% to nearly 100% in these cell lines, expressing a variety of thalassemic pre-mRNAs. The U7.623 lentivirus was then used to transduce blood cells from an individual with thalassemia carrying the IVS2-623 mutation with a 23-fold increase in corrected mRNA, providing proof of principle in human cells in vitro. Although correction rates were considerably lower in the IVS2-654 cell line, the availability of a transgenic mouse carrying the thalassemic 654 mutation should prove invaluable in further refining this clever strategy.