Scaffold attachment region-mediated enhancement of retroviral vector expression in primary T cells

Abstract

We have studied retroviral transgene expression in primary human lymphocytes. Our data demonstrate that transgene expression is high in activated primary CD4+ T cells but significantly decreased in mitotically quiescent cells. Incorporation of a DNA fragment from the scaffold attachment region (SAR) of the human beta interferon gene into the vector improved transgene expression, particularly in quiescent cells. The SAR element functioned in an orientation-dependent manner and enhanced expression of Moloney murine leukemia virus- and murine embryonic stem cell-based vectors. Clonal analysis of transduced T cells showed that the SAR sequence did not confer position-independent expression on a transgene but rather prevented the decrease of expression when cells became quiescent. The SAR sequence also enhanced transgene expression in T cells generated from retrovirally transduced CD34-enriched hematopoietic progenitor-stem cells in a SCID-hu thymus-liver mouse model. We have used the SAR-containing retroviral vector to express the RevM10 gene, a trans-dominant mutant of the human immunodeficiency virus type 1 (HIV-1) Rev gene. Compared to a standard retroviral vector, the SAR-containing vector was up to 2 orders of magnitude more efficient in inhibiting replication of the HIV-1 virus in infected CD4+ peripheral blood lymphocyte populations in vitro. This is the first demonstration that SAR elements can be used to improve retroviral vector expression in human primary T cells.

FIG. 1

Schematic representation of the retroviral vector constructs (not drawn to scale). The arrow above the SAR box indicates the orientation of the element. The small black box in the MESV-MiLy vector series indicates that the primer binding site is derived from the dl587rev retrovirus (9). Abbreviations: M10, RevM10 gene; MoMLV, MoMuLV LTR; MESV, MESV LTR; SAR, IFN-β SAR; IRES-Lyt2, internal ribosomal entry site linked to the Lyt-2 gene.

FIG. 2

The LMiLy2S vector is efficiently expressed in resting T cells. Lyt-2-enriched LMiLy- and LMiLy2S-transduced CD4⁺ primary T cells were activated with PHA, IL-2, and irradiated allogenic feeder cells. On days 3 and 11 poststimulation, cell aliquots were stained with anti-CD25 FITC-conjugated and anti-Lyt-2 PE-conjugated antibodies and analyzed by FACscan. The numbers indicate the percentage of Lyt-2⁺ cells in the respective quadrants. Gates for background fluorescence were set based on control isotype antibodies. Mock, untransduced control cells.

FIG. 3

Analysis of LMiLy2S vector expression in individual primary T-cell clones. Ten LMiLy2S (no. 1 through 10) and eight LMiLy (no. 11 through 18) T-cell clones were analyzed for Lyt-2 expression on days 3 (activated) and 11 (resting) after stimulation with PHA, IL-2, and irradiated allogenic feeder cells.

FIG. 4

The SAR effect is orientation dependent. (A) Lyt-2-enriched CD4⁺ primary T cells transduced with the MESV-MiLy, MESV-MiLy2S, MESV-MiLy2S-F, LMiLy, and LMiLy2S vectors were stimulated with PHA, IL-2, and feeder cells. Transgene expression was analyzed on days 3, 5, 7, 10, and 12 poststimulation as described in the legend to Fig. 2. On day 12, the cells were restimulated (indicated by arrow) and analyzed 3 days later (day 15 on the graph). (B) The percentage of Lyt-2⁺ cells in the CD25⁺ and CD25⁻ fractions of resting T cells was determined on day 10 poststimulation. Results of a representative experiment are shown. Results were reproduced with two separate tissues.

FIG. 5

Analysis of Lyt-2 transgene expression in thymocytes from HSPC-reconstituted SCID-hu thymus-liver grafts. (A) Mobilized peripheral blood CD34⁺ HSPCs were transduced with the LMiLy and LMiLyS vectors and analyzed for Lyt-2 expression 2 days posttransduction. (B) At 8 weeks after transplantation of SCID-hu thymus-liver mice with Lyt-2-enriched HSPC, thymus-liver grafts were harvested and freshly isolated thymocytes were examined for surface Lyt-2 (transgene) expression on donor HLA-positive cells (MA2.1⁺ thymocytes in a MA2.1⁻ host background). Percentages of Lyt-2⁺ cells were determined after subtraction of the background noise observed in mock-transduced reconstituted controls. Numbers (#) above FACS plots are as in Table 3 and indicate the individual SCID-hu animals. MF, relative Lyt-2 mean fluorescence intensity.

FIG. 6

HIV-1 infection experiment. Primary T cells were harvested on day 5 after stimulation with PHA, IL-2, and feeder cells and then inoculated with the HIV-1 JR-CSF virus. Viral replication was monitored for 9 days by measuring the p24 antigen concentration in cell supernatants. “Fully activated” samples (A) were supplemented with fresh PHA, IL-2, and feeder cells on day 3 after inoculation with HIV-1 to maintain activation of T cells, whereas “partially activated” samples (B) were maintained in medium with IL-2 only. All values are averages from triplicate samples; vertical bars indicate the standard errors (where not shown the error value was below the resolution of the graphics program). Results from a representative experiment are shown. Results were reproduced with two separate tissues.