A genetic trick used by viruses to replicate themselves has been adapted for laboratory use to build complex protein structures required by immune system cells, according to investigators at St. Jude Children’s Research Hospital.
This approach could also be used to develop new gene therapy vectors in cases when cells must be modified to make high levels of different proteins. A vector is a DNA molecule used to ferry specific genes into cells in order to give those cells the ability to make particular proteins.
A report on this work appears in the May 2004 issue of Nature Biotechnology.
The achievement gives researchers a powerful tool for studying the roles of complex proteins in living cells. The study also showed that this technique can reliably produce therapeutically useful amounts of multiple proteins. Some cellular proteins must be present in many copies in order to work efficiently. Because it only borrows a genetic trick from viruses but does not cause a real infection, the technique may increase the usefulness of current gene therapy vectors. Specifically, the technique would permit scientists either to restore complex protein structures that are missing in certain cells or make multiple proteins that act together as potent drugs against cancer and other diseases.
The technique is based on a genetic trick, called a self-cleaving 2A peptide, which is used by some viruses to produce multiple proteins from a single length of DNA; i.e., a single, long protein is produced that automatically breaks into multiple, distinct proteins.
St. Jude researchers used genetically modified mouse immune system cells called T lymphocytes to test the efficiency of this technique in making the CD3 complex, which is part of the T cell receptor, a large protein lodged in the cell’s membrane. The receptor allows T cells to “sense” targets that the cells are programmed to destroy. Without the CD3 complex, the T cell receptor is incomplete and cannot perform its immune function.
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.”