Highly efficient gene transfer in naive human T cells with a murine leukemia virus-based vector

Abstract

Retroviral vectors based on the Moloney murine leukemia virus (MuLV) have become the primary tool for gene delivery into hematopoietic cells, but clinical trials have been hampered by low transduction efficiencies. Recently, we and others have shown that gene transfer of MuLV-based vectors into T cells can be significantly augmented using a fibronectin-facilitated protocol. Nevertheless, the relative abilities of naive (CD45RA+) and memory (CD45RO+) lymphocyte subsets to be transduced has not been assessed. Although naive T cells demonstrate a restricted cytokine profile following antigen stimulation and a decreased susceptibility to infection with human immunodeficiency virus, it was not clear whether they could be efficiently infected with a MuLV vector. This study describes conditions that permitted gene transfer of an enhanced green fluorescent protein-expressing retroviral vector in more than 50% of naive umbilical cord (UC) blood and peripheral blood (PB) T cells following CD3/CD28 ligation. Moreover, treatment of naive T cells with interleukin-7 resulted in the maintenance of a CD45RA phenotype and gene transfer levels approached 20%. Finally, it was determined that parameters for optimal transduction of CD45RA+ T cells isolated from PB and UC blood differed: transduction of the UC cells was significantly increased by the presence of autologous mononuclear cells (24.5% versus 56.5%). Because naive T cells harbor a receptor repertoire that allows them to respond to novel antigens, the development of protocols targeting their transduction is crucial for gene therapy applications. This approach will also allow the functions of exogenous genes to be evaluated in primary nontransformed naive T cells.

Interleukin‐7 signalling is sufficient to phenotypically and functionally prime human CD4+ naïve T cells

“…The impact of IL‐7 on the phenotype of naïve T cells is controversial. Some studies have reported that IL‐7 does not alter the phenotype of naïve lymphocytes and has no effect on activation markers 39,46–48 . Others have observed that IL‐7 induces the up‐regulation of certain activation markers (CD25/CD95) but because these changes were not accompanied by conversion of the cells from CD45RA + to CD45RO + phenotype, these changes were classified as minimal 40,41 .…”

The impact of retroviral suicide gene transduction procedures on T cells

“…The effect of retroviral transduction on CD45RA and CD45RO T cells has not been extensively studied. Dardalhon et al (2000) used a retroviral vector encoding eGFP and packaged with GALV to transduce mature PBMC and T cells from umbilical cord collections. Naive umbilical cord T cells were shown to switch from CD45RA to the CD45RO phenotype after 6 d of stimulation, and were transduced at lower efficiency than mature T cells.…”

Use of suicide gene‐expressing donor T‐cells to control alloreactivity after haematopoietic stem cell transplantation

“…Optimization of culture conditions might contribute to increasing subsequent T‐cell function. We and others have observed that replacing OKT3/IL‐2 by CD3/CD28 at day 0 of cell culture might not only improve transduction efficiency [30] but also cell output [31], TCR diversity [29] and the frequency of anti‐EBV gene‐modified CD8 + T‐cells (Sauce et al ., submitted).…”

“…By increasing the polyclonality of GMC as well as reducing ex viv o culture time length, higher transduction efficiency might also contribute to enhancing overall immune function. Recently, technical improvements including use of Gibbon ape leukaemia virus enveloping pseudo‐typed retroviral vectors, CD3/CD28 costimulation, use of fibronectin‐coated plates as well as per‐transduction centrifugation have indeed resulted in improving transduction efficiency [30, 32–34].…”