Background: the Use of 2A in Human Gene Therapies. Until relatively recently gene therapies focussed upon repair of single gene lesions, due to the great difficulties associated with efficient co-expression of multiple proteins. Many gene therapies require, however, the co-ordinated expression of multiple genes. This may arise from the need to co-express the different chains of a hetero-multimeric protein, more than one transgene, or, therapeutic transgene(s) along with selectable markers (e.g. ex vivo gene therapy). Two major considerations are; (i) to ensure the efficient co-expression of the multiple transgenes within each individual cell and, (ii) minimisation of the number of chromosomal integration events thereby reducing the chance of harmful effects arising from ‘collateral’ genetic damage.

We discovered and developed a technology that allows multiple, different, proteins to be co-expressed (as discrete translation products) from a single mRNA. 2A and ‘2A-like’ sequences are short oligopeptides (~20aa) that mediate a co-translational ‘cleavage’ at their own C-termini. They can be used as linkers to concatenate multiple genes into a single, long, open reading frame (ORF). Translation of the single ORF produces each component of the polyprotein as an individual, discrete, product. Furthermore, co-translational signal sequences can be included immediately downstream of 2A within these artificial ‘self-processing’ polyprotein systems to target different components to different sub-cellular sites.

2A 'Cleavage' and Proteins Targeted to the Exocytic Pathway. We have discovered that there is a major problem with co-expression of some proteins targeted to, or transiting through, the endoplasmic reticulum (ER), leading to much lower levels of ‘cleavage’ (formation of fusion proteins) and hence aberrant sub-cellular localisation. Since ~39% of human proteins initially undergo co-translational transport through the ER membrane, many therapeutic strategies will require the co-expression of such proteins (as individual, discrete, translation products) with other proteins (targeted to different sub-cellular sites), selectable markers, or both.

The Aims of This Project are essentially twofold; (i) to more fully characterise the molecular basis of the inhibition of the 2A 'cleavage' reaction by such proteins transiting the ER membrane through the translocon pore and (ii) to harness this konwledge to provide optimal solutions for biomedical researchers involved in the development of new human gene therapies.