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Correction of multi-gene deficiency in vivo using a single 'self-cleaving' 2A peptide–based retroviral vector

Nature Biotechnology volume 22, pages 589–594 (2004)Cite this article

An Addendum to this article was published on 01 December 2004

A Corrigendum to this article was published on 01 June 2004

An Addendum to this article was published on 01 June 2004

Abstract

Attempts to generate reliable and versatile vectors for gene therapy and biomedical research that express multiple genes have met with limited success. Here we used Picornavirus 'self-cleaving' 2A peptides, or 2A-like sequences from other viruses1,2,3, to generate multicistronic retroviral vectors with efficient translation of four cistrons. Using the T-cell receptor:CD3 complex as a test system, we show that a single 2A peptide–linked retroviral vector can be used to generate all four CD3 proteins (CD3ε, γ, δ, ζ), and restore T-cell development and function in CD3-deficient mice. We also show complete 2A peptide–mediated 'cleavage' and stoichiometric production of two fluorescent proteins using a fluorescence resonance energy transfer–based system in multiple cell types including blood, thymus, spleen, bone marrow and early stem cell progenitors.

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Figure 1: Construction and processing of 2A-linked TCR:CD3 constructs.
Figure 2: Efficient cleavage and expression of TCR:CD3 complexes on 293T and 3T3 cells using 2A-linked vectors.
Figure 3: T-cell reconstitution in mice lacking all four CD3 molecules using a single, 2A-linked, multicistronic retroviral vector.
Figure 4: FRET analysis reveals efficient cleavage and stoichiometric expression of proteins generated from multicistronic 2A peptide–linked vectors in vivo.

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Acknowledgements

We are very grateful to Cox Terhorst and David Baltimore for reagents and Terry Geiger for his critical review of the manuscript. We also wish to thank Richard Cross, Jennifer Hoffrage, Richard Ashmun and Ann-Marie Hamilton-Easton for assistance with flow cytometry, staff in the Hartwell Center for oligo synthesis and DNA sequencing, and members of the Vignali lab for constructive criticism, comments and bone marrow isolation. This work was supported by grants from the National Institutes of Health (AI39480, AI52199), a Cancer Center Support CORE grant (CA-21765) and the American Lebanese Syrian Associated Charities to D.A.A.V.

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Author notes

  1. Elio F Vanin

    Present address: Center for Cell and Gene Therapy, Baylor College of Medicine, 6621 Fannin Street, Houston, Texas, 77030, USA

Authors and Affiliations

  1. Department of Immunology, St. Jude Children's Research Hospital, 332 N. Lauderdale, Memphis, 38105, Tennessee, USA

    Andrea L Szymczak, Creg J Workman, Yao Wang, Kate M Vignali, Smaroula Dilioglou & Dario A A Vignali

  2. Division of Experimental Hematology, Department of Hematology/Oncology, St. Jude Children's Research Hospital, 332 N. Lauderdale, Memphis, 38105, Tennessee, USA

    Elio F Vanin

  3. Graduate Program in Pathology, University of Tennessee Medical Center, 62 S. Dunlap, Memphis, 38163, Tennessee, USA

    Andrea L Szymczak

  4. Department of Pathology, University of Tennessee Medical Center, 62 S. Dunlap, Memphis, 38163, Tennessee, USA

    Andrea L Szymczak & Dario A A Vignali

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  1. Andrea L Szymczak
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Correspondence to Dario A A Vignali.

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Szymczak, A., Workman, C., Wang, Y. et al. Correction of multi-gene deficiency in vivo using a single 'self-cleaving' 2A peptide–based retroviral vector. Nat Biotechnol 22, 589–594 (2004). https://doi.org/10.1038/nbt957

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