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Advances
Published online: 12 April 2004 | doi:10.1038/nmeth009

Gene transfer

Put All Your Genes in One Basket

Michael Eisenstein
An unusual viral mechanism for the generation of multiple discreet peptides during the translation of a single mRNA transcript has given researchers a powerful new tool for coordinating the expression of multiple gene products from an individual recombinant retroviral construct, with potentially important repercussions for basic research and gene therapy studies.

For researchers wishing to express multiple genes from a single construct, the options are limited. The use of multiple promoters or internal ribosomal entry sites (IRES) can enable the expression of multiple, sequentially placed cistrons; however, there are a number of physical constraints on this system, and the level of expression for the various products will often vary substantially.

A new potential solution to this quandary appears in a recent article from Nature Biotechnology, presenting the work of Dario Vignali and his colleagues at St. Jude Children's Research Hospital (Memphis, TN). Vignali's group used specialized viral sequences to trigger the effective, non-enzymatic generation of several distinct peptide products from a single multicistronic construct.

According to Vignali, his team originally developed this system as the means to solve a particular research problem that they had encountered. "It was our work on the T-cell receptor that initiated our interest in 2A peptides. The T-cell receptor is a large, multimeric complex that contains the T-cell receptor dimmer and four CD3 molecules. All six chains have to be present for efficient cell surface expression. This makes reconstitution studies in mice very challenging. Instead of trying to express CD3 in four separate retroviral vectors, we wanted to find a way to put them all in one vector." He credits the virology research of Martin Ryan, of the University of St. Andrews (St. Andrews, Scotland), with first bringing his attention to the possibilities of the 2A-based 'self-cleavage' system.

Several viruses are known to use 2A peptide cleavage mechanisms. In vivo, this process results in the production of separate 2A and 2B peptides from a single transcript. Although the mechanism is loosely termed 'self-cleavage', it is understood that the 2A peptide actually contributes to a mechanism of 'ribosome skipping', wherein structural elements of the 2A fold impair the formation of a peptide bond between the terminal glycine of 2A and the initial proline of 2B. By transplanting the 2A consensus sequence in between two cistrons of interest, similar de facto cleavage can be obtained with recombinant gene expression constructs.

Vignali's group developed a variety of retroviral constructs incorporating various combinations of the TCR:CD3 chains separated by different 2A sequences. All the chains were expressed strongly, and the efficiency of cleavage approached 100% for all constructs tested. Additional fluorescence resonance energy transfer (FRET) experiments with 2A-linked YFP and CFP confirmed that the cleavage was efficient and stoichiometric. In cell culture experiments, functional TCRs were identified at the cell-surface following cotransduction with two retroviruses expressing all six TCR component peptides between them.

Equally encouraging results were seen with in vivo experiments to reconstitute TCR expression in CD3-deficient mice. Vignali's group transduced bone marrow from these mice with a retroviral construct containing the four missing CD3, and used this as a transplant substrate for irradiated T-cell-deficient recipients. T-cell reconstitution in these recipients approached levels seen following transplantation with wild-type bone marrow, and T-cells expressing the retroviral construct proliferated normally in response to immune stimulation.

Vignali is encouraged by the findings of these studies, both as a means for furthering his team's own T-cell investigations, and also for their potential contribution to a broad range of research fields. "We're not really in the business of developing novel gene therapy vectors," he says, "but we were so enamored with this system, we felt that it could be very useful for lots of different investigators. Not just those studying gene therapy, but also scientists wanting to express multiple proteins for their own basic research. I think that both basic and applied researchers will find many uses for this technology."

Published online 12 April 2004.

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REFERENCES
  1. Szymczak, A.L., Workman, C.J., Wang, Y., Vignali, K.M., Dilioglou, S., Vanin, E.F., and Vignali, D.A.A. Correction of Multi-Gene Deficiency in vivo Using a Single 'Self-Cleaving' 2A Peptide-Based Retroviral Vector. Nat Biotechnol. Online Publication, (4 April 2004).
  2. Donnelly, M.L., Luke, G., Mehrotra, A., Li, S., Hughes, L.E., Gani, D., and Ryan, M.D. Analysis of the Aphthovirus 2A/2B Polyprotein 'Cleavage' Mechanism Indicates Not a Proteolytic Reaction, but a Novel Translational Effect: a Putative Ribosomal 'Skip'. J. Gen. Virol. 82, 1013–1025 (2001). | PubMed | ISI | ChemPort |
  3. Donnelly, M.L., Hughes, L.E., Luke, G., Mendoza, H., ten Dame, E., Gani, D., and Ryan, M.D. The 'Cleavage' Activities of Foot-and-Mouth Disease Virus 2A Site-Directed Mutants and Naturally Occurring '2A-Like' Sequences. J. Gen. Virol. 82, 1027–1041 (2001). | PubMed | ISI | ChemPort |
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EISSN: 1548-7105
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