SMaRT for Therapeutic Purposes

Abstract

Spliceosome-mediated mRNA trans-splicing (SMaRT) is a promising strategy for treatment of genetic diseases which cannot be targeted via classical therapy approaches. SMaRT utilizes an exogenous pre-mRNA trans-splicing molecule (PTM) to correct a diseased target pre-mRNA. This process relies on splicing of two separate pre-mRNA molecules in trans creating a mature chimeric mRNA molecule which consists of the protein coding sequence of the PTM as well as the endogenous mRNA. For therapeutic implications, the most critical step in SMaRT is to develop PTMs resulting in a high ratio of trans-splicing to regular cis-splicing. This protocol provides guidelines on how to design PTMs and describes a fast screening assay to test their efficiencies. To elucidate the therapeutic potential of the best candidates in a more native setting, these PTMs are tested further on mini genes.