Retrovirus-mediated gene transfer in primary T lymphocytes impairs their anti–Epstein-Barr virus potential through both culture-dependent and selection process–dependent mechanisms

Functional impairment of human T-lymphocytes following PHA-induced expansion and retroviral transduction: implications for gene therapy

“…Functional analysis of protocols of polyclonal stimulation has been very limited so far, and focused on the function of CD3-stimulated GM T cells in vitro compared with CD3/CD28. 4,12 Despite PHA being a common polyclonal stimulus for transduction 2,6,13,14,16 and recent clinical data showing that the function of PHA-expanded GM T cells is poor in vivo compared with their unmanipulated counterparts, 16 no such comparative functional analysis has been published for PHA. In this study, we have explored the functional effect of PHA on T cells in vitro, compared it with iCD3/iCD28 stimulation, and then investigated the mechanisms leading to this functional effect.…”

“…12,15 To address this functional issue, we extended our analysis to explore the capacity of PHAexpanded T cells to mount antigen-specific responses against immunodominant peptides derived from Epstein-Barr virus (EBV) and cytomegalovirus (CMV). Dendritic cells (DCs) were generated from monocytes derived from CMV+/HLA-A2 and EBV+/HLA-B8 donors.…”

“…In fact, there is evidence to suggest that the expansion process rather than the integration or expression of the transgene may have the largest effect on the function of GM T cells in vitro and in animal models. 2,4,6,[9][10][11][12][13] In humans, although the data available are still limited, a number of trials showed that GM T cells can have reduced function in vivo. [14][15][16] Phytohemagglutinin (PHA) is a common polyclonal stimulus used in protocols of retroviral transduction.…”

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

“…In two constructs we dissected the V5 into two parts and accordingly made the two constructs as depicted in Figure 3e (second and third images). Next, we replaced the V5 peptide with a synthetic hinge region commonly used in chimeric TCR constructs 30 ((G4S)2, construct 4) or naturally occurring hinge regions from primate immunoglobulins 31 (constructs [5][6][7][8]. The original vector with the furin site plus the complete V5 peptide (construct 9) consistently displayed the highest level of TCR expression, whereas other vectors showed a performance similar to the vector where furin was linked directly to F2A (construct 10).…”

“…Furthermore, the current g-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-g 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 g-retroviral vectors have a limited coding capacity (o7 kb).…”