RNA Trans-Splicing Targeting Endogenous ?-Globin Pre-Messenger RNA in Human Erythroid Cells.

ABSTRACT: Sickle cell disease results from a point mutation in exon 1 of the ?-globin gene (total 3 exons). Replacing sickle ?-globin exon 1 (and exon 2) with a normal sequence by trans-splicing is a potential therapeutic strategy. Therefore, this study sought to develop trans-splicing targeting ?-globin pre-messenger RNA among human erythroid cells. Binding domains from random ?-globin sequences were comprehensively screened. Six candidates had optimal binding, and all targeted intron 2. Next, lentiviral vectors encoding RNA trans-splicing molecules were constructed incorporating a unique binding domain from these candidates, artificial 5' splice site, and ?-globin cDNA, and trans-splicing was evaluated in CD34+ cell-derived erythroid cells from healthy individuals. Lentiviral transduction was efficient, with vector copy numbers of 9.7 to 15.3. The intended trans-spliced RNA product, including exon 3 of endogenous ?-globin and ?-globin, was detected at the molecular level. Trans-splicing efficiency was improved to 0.07-0.09% by longer binding domains, including the 5' splice site of intron 2. In summary, screening was performed to select efficient binding domains for trans-splicing. Detectable levels of trans-splicing were obtained for endogenous ?-globin RNA in human erythroid cells. These methods provide the basis for future trans-splicing directed gene therapy.

SUBMITTER: Uchida N 

PROVIDER: S-EPMC5397226 | biostudies-literature | 2017 Apr

REPOSITORIES: biostudies-literature