Optimisation of the 2A Co-expression System for Gene Therapies

Gene therapy supplies your cells with genes either to replace faulty ones, or to supply genes that can combat other forms of disease. These genes are then translated into proteins. Often, however, a number of different proteins (and hence genes) are required for successful therapy. This work is designed to develop a system whereby multiple genes can be strung together to form a single gene: a multi-gene. It is much easier and safer to introduce one gene via gene therapy, than try to introduce many individual genes into a cell. The chances of this new type of multi-gene being able to produce all of its different proteins (and all at the same time) is very much higher than is the case with many individual genes. The methods we wish to develop here will extend the range of diseases that may be treated by gene therapy.

2A and 2A-like sequences are short, oligopeptide, sequences that mediate a co-translational cleavage at their own C-termini: they are used in gene therapy and other biomedical applications to concatenate multiple cistrons into a single open reading frame. Multiple proteins may, therefore, be co-expressed using from single transgene. We have discovered that sequences immediately upstream of 2A may inhibit the cleavage of proteins targeted to the exocytic pathway. Since ~40% of human proteins are (at least initially) translocated into the ER, gene therapies will inevitably involve the co-expression of such proteins. Our data strongly suggest an interaction between the nascent protein and the translocon pore is responsible for this inhibition. This application seeks to perform a fine mapping of the inhibitory interactions and through a program of screening site-directed mutants/insertions to identify methods of achieving high-level 2A-mediated cleavage of such proteins. The partition of proteins (derived from 2A-mediated cleavage)between the exocytic pathway and the cytoplasm will be measured to compare the the relative efficiencies of different 2A-linker sequences. The aim is to develop the technology to provide gene therapists with a complete tool-kit for protein co-expression.