biotech>>genetics
The genetic engineering of human lymphocytes as a potential therapy for inherited, acquired or infectious disease requires efficient transfer and expression of the transgene. In the case of adoptive immunotherapy for cancer, naturally occurring antitumor T-cell receptors (TCRs) have been used to endow normal T cells with antitumor reactivity.1 In a recent clinical trial, we demonstrated the successful conferral of antitumor function through ex vivo -retroviral transduction of a MART-1-reactive TCR gene into the peripheral blood lymphocytes (PBLs) in patients with metastatic melanoma resulted in clinical tumor responses.2 Although successful in demonstrating the potential of this cancer gene therapy approach, there are several limitations to current -retroviral vector-based gene transfer systems.
In T-cells, the level of transgene expression is modulated by the activation state of the cell,3 and -retroviral based vectors have been shown to be subject to gene silencing in certain cell types,4, 5 and they have a preference for integration near transcription start sites,6 which may increase the potential for insertional mutagenesis. Furthermore, the current -retroviral vector based protocols require T cells to be fully activated for efficient transduction and this may be deleterious to their function.7, 8 We previously reported that in a murine model, prolonged ex vivo stimulation yielded fully differentiated effector T cells which were capable of in vitro tumor killing and high-level INF- release but, exhibited inferior activity for in vivo tumor treatment compared to naive and less differentiated effectors.9 Finally, there is a growing need for gene delivery systems to carry multiple genes in one construct, and -retroviral vectors have a limited coding capacity (<7 kb).
Lentiviral vectors with their larger coding capacity and ability to more easily express complex expression cassettes may be advantageous in this regard compared to -retroviral vectors.10, 11 An example of a clinically important multi-gene construct is a tumor-associated antigen TCR, which is a heterodimer of TCR- and - chains.12 The TCRs for a variety of tumor-associated antigens (TAA) have been identified including the MART-1 and gp100 melanoma differentiation antigens, the NY-ESO-1 cancer-testis antigen and the p53 tumor suppressor.13, 14, 15, 16, 17, 18 An important aspect of TCR expression vector design is that, the TCR-- and - chains must be coordinately expressed for proper biologically activity.12
The most common strategy for the expression of multiple genes has been based on internal ribosome entry site (IRES) elements.19 However, bicistronic lentiviral vectors containing IRES elements have consistently demonstrated a biased expression of two transgenes with the second gene being under expressed.20, 21, 22 We confirmed these reports (data not shown) and thus assembled a series of dual promoter-containing lentiviral vectors to express the - and -TCR chains, but consistently failed to achieve a high percentage of TCR expression in PBL (S Jones, data not shown). Naldini and co-workers recently reported that lentiviral vectors coordinately expressing two genes could be assembled using a synthetic bidirectional promoter,21 but this synthetic promoter exhibited poor activity in PBL (approximately 5–10% of transduced cells expressed both genes). An alternative to these approaches is the use of ribosomal skipping via 2A peptides.23, 24 Several viruses use 2A peptides to mediate protein cleavage, including foot-and-mouth disease virus (F2A), equine Rhinitis A virus, porcine teschovirus-1 (P2A) and Thosea asigna virus (T2A). The 2A peptide consensus motif (DVEXNPGP) is extremely rare and is associated with cleavage-like activity between 2A and 2B genes through a ribosomal skip mechanism; the 2A peptide impairs normal peptide bond formation between 2A and 2B without affecting the translation of 2B. 2A peptides have been shown in -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- and - 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. Furin is a ubiquitous subtilisin-like proprotein convertase, whose natural substrates include certain serum proteins and growth factor receptors, such as the insulin-like growth factor receptor. The consensus sequence for furin cleavage is RXXR but the potential for actual cleavage is dependent on substrate tertiary structure and the amino acids immediately surrounding the recognition site.26
Herein, we developed a novel bicistronic lentiviral vector that combines a furin cleavage site, and an amino-acid spacer followed by a 2A ribosomal skip peptide. When the spacer sequence was augmented by the addition of a synthetic V5 peptide tag sequence, this specifically boosted protein processing and resulted in a lentiviral vector capable of mediating high-level TCR expression in transduced lymphocytes.
In T-cells, the level of transgene expression is modulated by the activation state of the cell,3 and -retroviral based vectors have been shown to be subject to gene silencing in certain cell types,4, 5 and they have a preference for integration near transcription start sites,6 which may increase the potential for insertional mutagenesis. Furthermore, the current -retroviral vector based protocols require T cells to be fully activated for efficient transduction and this may be deleterious to their function.7, 8 We previously reported that in a murine model, prolonged ex vivo stimulation yielded fully differentiated effector T cells which were capable of in vitro tumor killing and high-level INF- release but, exhibited inferior activity for in vivo tumor treatment compared to naive and less differentiated effectors.9 Finally, there is a growing need for gene delivery systems to carry multiple genes in one construct, and -retroviral vectors have a limited coding capacity (<7 kb).
Lentiviral vectors with their larger coding capacity and ability to more easily express complex expression cassettes may be advantageous in this regard compared to -retroviral vectors.10, 11 An example of a clinically important multi-gene construct is a tumor-associated antigen TCR, which is a heterodimer of TCR- and - chains.12 The TCRs for a variety of tumor-associated antigens (TAA) have been identified including the MART-1 and gp100 melanoma differentiation antigens, the NY-ESO-1 cancer-testis antigen and the p53 tumor suppressor.13, 14, 15, 16, 17, 18 An important aspect of TCR expression vector design is that, the TCR-- and - chains must be coordinately expressed for proper biologically activity.12
The most common strategy for the expression of multiple genes has been based on internal ribosome entry site (IRES) elements.19 However, bicistronic lentiviral vectors containing IRES elements have consistently demonstrated a biased expression of two transgenes with the second gene being under expressed.20, 21, 22 We confirmed these reports (data not shown) and thus assembled a series of dual promoter-containing lentiviral vectors to express the - and -TCR chains, but consistently failed to achieve a high percentage of TCR expression in PBL (S Jones, data not shown). Naldini and co-workers recently reported that lentiviral vectors coordinately expressing two genes could be assembled using a synthetic bidirectional promoter,21 but this synthetic promoter exhibited poor activity in PBL (approximately 5–10% of transduced cells expressed both genes). An alternative to these approaches is the use of ribosomal skipping via 2A peptides.23, 24 Several viruses use 2A peptides to mediate protein cleavage, including foot-and-mouth disease virus (F2A), equine Rhinitis A virus, porcine teschovirus-1 (P2A) and Thosea asigna virus (T2A). The 2A peptide consensus motif (DVEXNPGP) is extremely rare and is associated with cleavage-like activity between 2A and 2B genes through a ribosomal skip mechanism; the 2A peptide impairs normal peptide bond formation between 2A and 2B without affecting the translation of 2B. 2A peptides have been shown in -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- and - 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. Furin is a ubiquitous subtilisin-like proprotein convertase, whose natural substrates include certain serum proteins and growth factor receptors, such as the insulin-like growth factor receptor. The consensus sequence for furin cleavage is RXXR but the potential for actual cleavage is dependent on substrate tertiary structure and the amino acids immediately surrounding the recognition site.26
Herein, we developed a novel bicistronic lentiviral vector that combines a furin cleavage site, and an amino-acid spacer followed by a 2A ribosomal skip peptide. When the spacer sequence was augmented by the addition of a synthetic V5 peptide tag sequence, this specifically boosted protein processing and resulted in a lentiviral vector capable of mediating high-level TCR expression in transduced lymphocytes.
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