343. Repair of Thalassemic β-Globin pre-mRNA by Antisense AAV and Lentiviral Vectors in Cell Culture

Abstract

b-Thalassemia is a worldwide genetic blood disorder characterized by a deficiency or lack of the b-globin chain production. More than 200 mutations in the b-globin gene are responsible for this disease. Several common mutations create aberrant splice sites in the b-globin pre-mRNA, leading to an incorrectly spliced b-globin mRNA. Previous study in this lab showed that a modified U7.623 snRNA, which carries a specific sequence antisense to the splicing enhancer sequence of the three b-thalassemic mutants (IVS2-654, -705, and -745), could modify the splicing pathway and restore correctly spliced b-globin mRNA and protein in cell culture. In this study we incorporated the U7.623 antisense snRNA into lentiviral- and AAV-vectors. Transient transfection of HeLa cell lines expressing IVS2-654, and-705, with an AAV-U7 antisense snRNA plasmid, resulted in a restoration of both correctly spliced b-globin mRNA and full-length b-globin polypeptide. The results show that antisense snRNAs shifted the splicing pathway from aberrant to correct and restored the correct mRNAs in a sequence specific and dose-dependent manner. This was accomplished in model cell lines, in which the thalassemic b-globin intron was incorporated into the enhanced green fluorescence protein (EGFP) coding sequence and in HeLa cells that express thalassemic human b-globin genes. Transfection of EGFP-HeLa cell lines with IVS2-654, and -705U introns with AAV-U7.623 resulted in upregulation and expression of EGFP protein concomitant with expression of the of red fluorescence protein (RFP), an AAV viral reporter gene. Similar results, although less pronounced, were obtained in cells transiently transfected with lentiviral-U7.623 antisense snRNA construct. Interestingly, the restoration of correctly spliced mRNAs was faster in AAV than lentiviral vector treated cells. Based on this encouraging result, the AAV- and lentiviral-U7 antisense snRNA constructs will be evaluated in animal models for thalassemia. The snRNA antisense-based gene therapy may offer an attractive approach to cure thalassemia.