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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

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).

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 |

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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ISSN: 1548-7091
EISSN: 1548-7105

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