Advances in the field of lentivector-based transduction of T and B lymphocytes for gene therapy

Abstract

Efficient gene transfer into quiescent T and B lymphocytes for gene therapy or immunotherapy purposes may allow the treatment of several genetic dysfunctions of the hematopoietic system, such as immunodeficiencies, and the development of novel therapeutic strategies for cancers and acquired diseases. Lentiviral vectors (LVs) can transduce many types of nonproliferating cells, with the exception of some particular quiescent cell types such as resting T and B cells. In T cells, completion of reverse transcription (RT), nuclear import, and subsequent integration of the vesicular stomatitis virus G protein pseudotyped LV (VSVG-LV) genome does not occur efficiently unless they are activated via the T-cell receptor (TCR) or by survival-cytokines inducing them to enter into the G(1b) phase of the cell cycle. Lentiviral transduction of B cells is another matter because even B-cell receptor-stimulation inducing proliferation is not sufficient to allow efficient VSVG-LV transduction. Recently, a new LV carrying the glycoproteins of measles virus (MV) at its surface was able to overcome vector restrictions in both quiescent T and B cells. Importantly, naive as well as memory T and B cells were efficiently transduced while no apparent activation, cell-cycle entry, or phenotypic switch were detected, which opens the door to a multitude of gene therapy and immunotherapy applications as reported here.

Figure 1

Efficient transduction of quiescent T and B lymphocytes by measles virus glycoprotein pseudotyped LVs. Adult quiescent (a) T and (b) B cells were isolated from peripheral blood and immediately transduced with MV-LVs or VSVG-LVs at multiplicity of infection = 10 and 30, respectively. GFP expression was analyzed at day 3 after transduction by fluorescent-activated cell sorting (FACS). The percentage of GFP-positive cells is indicated in T cells in a (left plots) and for B cells in b (left plots). In parallel, cell-cycle progression was monitored 3 days post-transduction by simultaneously visualizing the RNA (Pyronine-Y) and DNA (7-AAD) content of the T cells (a, right dot plots) and B cells (b, right dot plots). The percentages of cells in the G₀/G_1a, G1_b, S/G₂/M phase of the cell cycle are indicated. Surface SLAM expression was assessed by FACS analysis before transduction by staining with anti-CD150-phycoerythrin. Percentage of SLAM expression is indicated in the histogram for (c) T and (d) B cells. 7-AAD, 7-amino-actinomycin D; GFP, green fluorescent protein; LV, lentiviral vector; MV, measles virus; VSVG, vesicular stomatitis virus G protein.

Figure 2

Schematic presentation of the important restrictive steps in lentiviral transduction of quiescent lymphocytes. VSVG-LVs are able to enter into quiescent T or B cells through a yet unknown ubiquitously expressed receptor. However, they show very low reverse transcription activity and nuclear import resulting in low proviral integration efficiency. This was only partially overcome by addition of nucleosides that increase reverse transcriptase (RT) activity and ATP increase to induce nuclear import. On the other hand, MV-LVs use the CD46 and SLAM receptors for entry into quiescent T and B cells. Upon entry, very rapidly a much more efficient RT activity was detected as compared to VSVG-LVs in these cells without affecting their quiescent state. This was followed by nuclear import and finally efficient proviral integration. LV, lentiviral vector; MV, measles virus; VSVG, vesicular stomatitis virus G protein.