An in vivo selection method to optimize trans-splicing ribozymes

Abstract

Group I intron ribozymes can repair mutated mRNAs by replacing the 39-terminal portion of the mRNA with their own 39-exon. This trans-splicing reaction has the potential to treat genetic disorders and to selectively kill cancer cells or virus-infected cells. However, these ribozymes have not yet been used in therapy, partially due to a low in vivo trans-splicing efficiency. Previous strategies to improve the trans-splicing efficiencies focused on designing and testing individual ribozyme constructs. Here we describe a method that selects the most efficient ribozymes from millions of ribozyme variants. This method uses an in vivo rescue assay where the mRNA of an inactivated antibiotic resistance gene is repaired by trans-splicing group I intron ribozymes. Bacterial cells that express efficient trans-splicing ribozymes are able to grow on medium containing the antibiotic chloramphenicol. We randomized a 59-terminal sequence of the Tetrahymena thermophila group I intron and screened a library with 9 3 10 6 ribozyme variants for the best trans-splicing activity. The resulting ribozymes showed increased trans-splicing efficiency and help the design of efficient trans-splicing ribozymes for different sequence contexts. This in vivo selection method can now be used to optimize any sequence in trans-splicing ribozymes.

Trans-splicing with the group I intron ribozyme from Azoarcus

“…The trans-splicing efficiencies of Azoarcus ribozyme constructs in E. coli cells were much lower than the trans-splicing efficiencies of Tetrahymena ribozymes in E. coli cells of 1%-10% (Sullenger and Cech 1994;Olson and Muller 2012) and this study. Similarly, no cis-splicing Azoarcus ribozyme construct mediated chloramphenicol resistance in E. coli cells or achieved cis-splicing efficiencies above 5%, whereas a cis-splicing Tetrahymena ribozyme construct mediated chloramphenicol resistance and achieved ∼60% cissplicing efficiency in cells (Fig.…”

“…The Tetrahymena ribozyme achieved up to ∼20% trans-splicing efficiency on full-length CAT mRNA with the "classical design," which includes a short P1 extension, an internal loop that may form a P10 helix, and a 5 ′ duplex with the substrate ( Fig. 3; Köhler et al 1999;Byun et al 2003;Einvik et al 2004;Lundblad et al 2004;Olson and Muller 2012). The Azoarcus ribozyme also achieved up to ∼20% trans-splicing efficiency but with the "anticodon stem design" (Fig.…”

“…The internal loop region of the EGS may also form a P10 duplex with the 3 ′ exon, which can benefit the transsplicing efficiency ( Fig. 3A; Köhler et al 1999;Byun et al 2003;Olson and Muller 2012). Subsequent studies found that this "classical design" also increases the transsplicing efficiencies of the group I introns from Fuligo and Didymium Fiskaa et al 2006).…”

“…The EGS is an elongation of the 5 ′ terminus of the trans-splicing ribozyme and has been tested on the group I intron ribozymes from Tetrahymena thermophila (subgroup IC1), Didymium (subgroup IE), and Fuligo (subgroup IC1) Byun et al 2003;Fiskaa et al 2006;Olson and Muller 2012). Despite the improvements mediated by the EGS, the transsplicing efficiency remains a limiting factor for potential therapeutic applications of these ribozymes (Fiskaa and Birgisdottir 2010).…”

Increased efficiency of evolved group I intron spliceozymes by decreased side product formation

“…2A) using an evolution system established in earlier studies (Olson and Muller 2012;Olson et al 2014). As starting points for the evolution, two spliceozyme libraries were generated by mutagenic PCR, using as template either a single spliceozyme gene ( Fig.…”