440. Optimization of RNA Trans-Splicing for the β-Globin Gene; Detecting Trans-Splicing Events Targeting Endogenous β-Globin Pre-mRNA in Human Erythroid Cells
Authors: Naoya Uchida, Charlotte Platner, Josiah Ballantine, Matthew M. Hsieh, Brian Mozer, Lloyd G. Mitchell, Kareem N. Washington, John F. Tisdale
Journal: Molecular Therapy (2015)

Sickle cell disease is caused by a point mutation in exon 1 of the β-globin gene (3 exons and 2 introns). RNA splicing between two distinct pieces of pre-mRNA, known as trans-splicing, represents a potential therapeutic strategy allowing replacement of the mutated exon 1 with a normal exon. Induction of exogenous mRNA splicing using spliceosome-mediated trans-splicing requires an RNA trans-splicing molecule (RTM) which imitates endogenous cis-splicing elements. However, clinical application of trans-splicing for globin disorders will require high efficiency, thus we sought to optimize the efficiency of trans-splicing targeting the β-globin gene to replace the exon 1 among human erythroid cells. To optimize the binding domain targeting the β-globin gene, the randomized binding domains of 20-600b were generated by sonicating the β-globin gene, and these were inserted into RTM-expressing plasmids downstream of the 5' half of GFP and a 5' splice site. In addition, we designed a target plasmid which encodes the β-globin gene and an artificial 3' splice site connected to 3' site of the other half of GFP. In transfection of both RTM and target plasmids, we selected 6 candidates from several thousand RTMs, which produced the brightest GFP-positive cells (maximal trans-splicing), and interestingly, all 6 binding domains targeted the β-globin intron 2. To evaluate efficiency of trans-splicing for human endogenous β-globin RNA, we constructed lentiviral vectors encoding RTMs which contain the γ-globin cDNA connected to a 5' splice site and 3 candidates (1E1; 0.6kb, 2D10; 0.7kb, and 13-1; 0.8kb) of β-globin binding domains (selected from the 6 candidates). Human CD34+ cells were transduced with these RTM-encoding lentiviral vectors, and these cells were differentiated to erythroid cells in vitro. Trans-splicing was detected by RT-PCR and sequencing in RTM 2D10 and 13-1 but not in RTM 1E1, suggesting that longer binding domains improve trans-splicing efficiency. To elongate the binding domain in RTM 13-1, we designed RTM BGin2 (containing 5' splice site of β-globin intron 2; 0.9kb) and BGex1-in2 (containing whole β-globin sequence except exon 3; 1.3kb). In both RTM BGin2 and BGex1-in2, ~4-fold higher trans-splicing was detected by RT-qPCR than RTM 13-1, which resulted in ~0.07% trans-splicing efficiency as compared to endogenous human β-globin RNA, implying that the 5' splice site of intron 2 should be included as a binding domain for efficient trans-splicing. In summary, we developed a screening system to select more efficient binding domains for trans-splicing. For the first time, we obtained detectable levels of spliceosome-mediated trans-splicing (~0.07%) for endogenous β-globin RNA in human erythroid cells which were transduced with RTM-expressing lentiviral vectors. Further optimization is required to develop trans-splicing based gene therapy.