Repair of CFTR mRNA by spliceosome-mediated RNA trans-splicing

Abstract

Most messenger RNA precursors (pre-mRNA) undergo cissplicing in which introns are excised and the adjoining exons from a single pre-mRNA are ligated together to form mature messenger RNA. This reaction is driven by a complex known as the spliceosome. Spliceosomes can also combine sequences from two independently transcribed pre-mRNAs in a process known as trans-splicing. Spliceosome-mediated RNA trans-splicing (SMaRT) is an emerging technology in which RNA pre-therapeutic molecules (PTMs) are designed to recode a specific pre-mRNA by suppressing cis-splicing while enhancing trans-splicing between the PTM and its premRNA target. This study examined the feasibility of SMaRT as a potential therapy for genetic diseases to correct mutations using cystic fibrosis (CF) as an example. We used several versions of a cystic fibrosis transmembrane conduc-

Correction of dystrophia myotonica type 1 pre-mRNA transcripts by artificial trans-splicing

“…Previously reported experiments using co-transfecting mini-gene targets and ATMs have also demonstrated high levels of RNA trans-splicing compared to endogenous premRNA trans-splicing, with efficiency ranging from 1 to 80% based on real-time RT-PCR detection. 12,26,27 However, the efficiency of trans-splicing to an endogenous pre-mRNA target expressed in a stable cell line has generally been lower; with reports of between 3 and 7% efficiency when assayed by PCR, 13,26 which is comparable to the maximum trans-splicing efficiency achieved in our study.…”

“…18 SMaRT has been shown to repair defective pre-mRNA molecules in cultured mammalian cells, xenografts and animal models of human disease including cystic fibrosis, 12 hemophilia A (factor VIII deficiency), 13 X-linked CD40 ligand immunodeficiency, 14 human apolipoprotein A1, 15 DNA protein kinase deficiency, 19 epidermolysis bullosa simplex with muscular dystrophy 20 and spinal muscular atrophy. 21 In DM1, a strategy to remove or correct mutant mRNA is appropriate in view of evidence that the accumulation of mutant mRNA in the nucleus is the primary basis of the disease.…”

“…The ATMs were constructed with an antisense-binding region of different lengths complementary to the 3 0 end of intron 14, 3 0 -splicing elements and the coding sequence of DMPK exon 15 fused in reading frame with GFP (see Materials and Methods, and Figure 1a). The construction of the ATMs was based primarily on the approach of Mansfield et al 12 who had designed two types of trans-splicing molecules, carrying linear and safety spacer sequences. Likewise, we constructed four linear ATMs (ATM130, ATM166, ATM214 and ATM250), four safety ATMs (ATM130S, ATM166S, ATM214S and ATM250S) and one negative control ATM without an antisense-binding region.…”

“…12 Four different lengths of antisense-binding domain regions were designed to target intron 14 and at least one of two predicted U2-binding domains. We predicted two U2-binding domains in DMPK intron 14, namely (CTAAC) located at À138 to À134 bp (PU2) and a reverse sequence complimentary to AGCCAATCGA located at À49 to À39 bp (PU2r) from exon 15.…”

“…We examined artificial trans-splicing of corrective molecules as a molecular therapeutic strategy for DM1, namely the technique of spliceosome-mediated RNA transsplicing (SMaRT; Intronn proprietary technology, http://www.intronn.com). [12][13][14][15][16][17] In this study, the goal is to develop a trans-splicing system for DM1 and to investigate the efficiency of artificial trans-splicing molecules (ATMs) to target and correct the DMPK transcript without degradation. Our approach provides intron-specific gene correction at the pre-mRNA level in the nucleus.…”