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, 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 (CD3epsilon, gamma, delta, zeta), 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.

Development of optimal bicistronic lentiviral vectors facilitates high-level TCR gene expression and robust tumor cell recognition

“…Encouraged by the successful use of 2A peptides in g-retroviral vectors to express multiple genes in one construct, 24,25,27 we attempted to develop lentiviral vectors expressing antitumor antigen-specific TCRs using different 2A peptides. In Figure 1a, the amino acids from different 2A peptides are illustrated and the conservative domains highlighted.…”

“…It has been reported that in g-retroviral constructs, expression of multiple genes linked with 2A peptides can be facilitated by a spacer sequence (GSG) ahead of the 2A peptides. 25,27 We designed several constructs combining a spacer (SGSG or GSG) and furin (R-A-K-R) 28 cleavage site with different 2A peptides in bicistronic lentiviral vectors (Figure 1b).…”

“…2A peptides have been shown in g-retroviral vectors to initiate the production of up to four proteins both in vitro and in vivo. 24,25 When exogenous genes are linked using this approach, about 18 amino acids are left on the C terminus of the first gene and a single proline residue remains on the N terminus of the second gene. Although linking of the TCR-a and -b chains by 2A peptide has been reported, 25 the effect of the residual amino acids on the function of expressed TCR has not been fully explored.…”

“…24,25 When exogenous genes are linked using this approach, about 18 amino acids are left on the C terminus of the first gene and a single proline residue remains on the N terminus of the second gene. Although linking of the TCR-a and -b chains by 2A peptide has been reported, 25 the effect of the residual amino acids on the function of expressed TCR has not been fully explored. In this study, we inserted a furin protease recognition site ahead of the 2A peptide to obtain a more native TCR chain by post-translational processing.…”

Mechanism and methods to induce pluripotency

“…To achieve simultaneous expression of reprogramming factors, cDNA encoding as many as five reprogramming factors connected by the 2A peptide sequence from the foot-and-mouth disease virus was cloned in a single vector. Individual proteins were produced through a "ribosomal skipping" mechanism mediated by the 2A peptide (Szymczak et al, 2004;Yusa et al, 2009). In the case of the piggyBac system, after successful generation of iPS cells, the reprogramming cassette can be removed by piggyBac transposase without footprint and transgene-free iPS cells were obtained (Yusa et al, 2009).…”