Using 5′-PTMs to repair mutant β-globin transcripts

Abstract

Trans-splicing has been used to repair mutant RNA transcripts via competition for the spliceosome using pre-trans-splicing molecules, or ''PTMs.'' Previous studies have demonstrated that functional PTMs can be designed for either 39-or 59-exon replacement, with a vast majority of the work to date focusing on repair of mutations within internal exons and via 39-exon replacement. Here, we describe the first use of trans-splicing to target the first exon and intron of a therapeutically relevant gene and repair the mutant RNA by 59-exon replacement. Our results show that 59-PTMs can be designed to repair mutations in the b-globin transcript involved in sickle cell anemia and b-thalassemia while providing insight into considerations for competition between trans-versus cis-splicing in mammalian cells. Target transcripts with impaired cis-splicing capabilities, like those produced in some forms of b-thalassemia, are more efficiently repaired via trans-splicing than targets in which cis-splicing is unaffected as with sickle b-globin. This study reveals desirable characteristics in substrate RNAs for trans-splicing therapeutics as well as provides an opportunity for further exploration into general splicing mechanisms via 59-PTMs.

High-Throughput Screening for Highly Functional RNA-Trans-Splicing Molecules: Correction of Plectin in Epidermolysis Bullosa Simplex

“…Such settings were reported for epidermolysis bullosa (COL7A1, KRT14, PLEC) (Murauer et al, 2010;Wally et al, 2010;Wally et al, 2008), Duchenne muscular dystrophy (Lorain et al, 2010), cystic fibrosis (Liu et al, 2002;Song et al, 2009), frontotemporal dementia with parkinsonism (Rodriguez-Martin et al, 2009), severe combined immunodeficiency (Zayed et al, 2007), spinal muscular atrophy (Coady et al, 2007), sickle cell anemia and ß-thalassemia (Kierlin-Duncan and Sullenger, 2007). First in vivo assays showed the functionality of SMaRT in a mouse model of spinal muscular atrophy (Coady et al, 2008;Coady and Lorson, 2010).…”

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

“…10,12 A later report indicated that ribozyme-mediated trans-splicing (*36%) was detected in 293T cells with co-transfection of a target plasmid (mutated b-globin intron 1 3¢ splice site) and ribozyme plasmid targeting the intron 1 (binding domain and b-globin). 11 Transsplicing is less efficient than normal cis-splicing, and reasonably high levels of trans-splicing efficiency are needed for clinical application.…”

“…In a cell line model with co-transfection of a target plasmid (b-globin gene) and an RTM plasmid (binding domain and c-globin cDNA), initially both trans-splicing events and b-globin expression were detected by the same RT-PCR reaction (data not shown), similar to previous reports using a ribozyme. [10][11][12] However, the ratios of DNA band densities between b-globin cDNA (target) and trans-splicing products were strongly affected by PCR annealing temperatures. To calculate the efficiency of trans-splicing precisely (trans-splicing events per b-globin RNA), a more reliable RT-qPCR method was established by using a 1:1 molecular ratio of control plasmid containing both the transsplicing sequence and the b-globin gene.…”

“…RNA splicing from two different pieces of premessenger RNA, known as trans-splicing, is another method, allowing the mutated exon 1 to be replaced with a normal exon. [10][11][12] Spliceosomemediated RNA trans-splicing would be induced by an RNA trans-splicing molecule (RTM), which contains (1) a coding region to replace mutation in target RNA, (2) a 5¢ or 3¢ splice site, and (3) a binding domain, which is complementary to target RNA. 13 A major benefit of the trans-splicing-based gene therapy over the whole gene insertion strategy in SCD is the ability to induce therapeutic gene expression and to reduce pathogenic sickle globin production concurrently.…”

“…The trans-splicing in b-globin RNA was previously reported in cell line models. [10][11][12] However, the clinical application for SCD will require high efficiency of trans-splicing. Therefore, this study sought to improve the efficiency of trans-splicing targeting the b-globin gene, which replaces exon 1 in human erythroid cells.…”