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

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 viruses^1,2,3, 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.