The St. Jude researchers used retroviral vectors as the delivery system into which they inserted cassettes (groups of genes) that contained genes for the four CD3 proteins, separated by the 2A peptides. These 2A peptides acted like cleavers to break apart the long protein into the four different, smaller CD3 proteins. The cell used these smaller proteins to build the large TCR:CD3 receptor. In order to replicate inside a cells, the retrovirus RNA must first be changed back into DNA. A retrovirus is a virus whose genetic material is RNA instead of DNA.

The St. Jude team used these multicistronic retroviral vectors (vectors carrying several different genes) to deliver the 2A peptide-linked CD3 gene cassettes into hematopoietic stem cells from mice that lacked the CD3 proteins, and thus could not make T cells. These genetically modified stem cells subsequently developed and restored T cell development in the mice. Hematopoietic stem cells are parent cells that give rise to all the red and white cells found in blood.

These 2A peptides will allow us, and others, to generate single vectors that can efficiently and reliably express multiple proteins in the exact amounts needed to permit the cell to assemble complex structures, said Dario A. A. Vignali, Ph.D., associate member of the St. Jude Department of Immunology and a faculty member at the University of Tennessee Medical Center.

Vignali is senior author of the Nature Biotechnology report.

We expect that this technique will make it a lot easier for us to study the role of complex protein structures, Vignali said. These 2A peptides may also facilitate the development of more versatile gene therapy vectors for treatments that require replacement or expression of more than a single gene.