A compact and simple method of achieving differential transgene expression by exploiting translational readthrough

The development of multicistronic vectors enabling differential transgene expression is a goal of gene therapy and poses a significant engineering challenge. Current approaches rely on the insertion of long regulatory sequences that occupy valuable space in vectors, which have a finite and limited packaging capacity. Here we describe a simple method of achieving differential transgene expression by inserting stop codons and translational readthrough motifs (TRMs) to suppress stop codon termination. TRMs reduced downstream transgene expression similar to sixfold to similar to 140-fold, depending on the combination of stop codon and TRM used. We show that a TRM can facilitate the controlled secretion of the highly potent cytokine IL-12 at therapeutically beneficial levels in an aggressive immunocompetent mouse melanoma model to prevent tumor growth. Given their compact size (6 bp) and ease of introduction, we envisage that TRMs will be widely adopted in recombinant DNA engineering to facilitate differential transgene expression. Method summary We describe a simple method enabling two or more gene products to be synthesized in a cell at different levels to aid in the development of new therapeutics.

Automated summary

This study describes a simple method of achieving differential transgene expression by inserting stop codons and translational readthrough motifs (TRMs). TRMs can reduce downstream transgene expression and facilitate the controlled secretion of cytokines at therapeutically beneficial levels to prevent tumor growth. Due to their compact size and ease of introduction, TRMs are expected to be widely adopted in recombinant DNA engineering.