Efficient transduction by an amphotropic retrovirus vector is dependent on high-level expression of the cell surface virus receptor

Abstract

Transduction by murine leukemia virus-based retrovirus vectors is limited in certain cell types, particularly in nondividing cells. But transduction can be inefficient even in cells that divide rapidly. For example, exposure of 208F rat embryo fibroblasts to an excess of an amphotropic retrovirus vector encoding alkaline phosphatase results in a transduction efficiency of only about 10%, even though these cells divide rapidly. Here we show that transduction of 208F cells is limited by cell surface retrovirus receptor levels; overexpression of the amphotropic retrovirus receptor Pit2 markedly improved the transduction efficiency to 50%. To characterize receptor levels and binding affinity, we synthesized a fusion protein that joins the amino terminus of the amphotropic envelope protein to the Fc region of a human immunoglobulin G1 molecule for use in binding assays. In comparison to the parental cell line, the modified cell line showed an order of magnitude increase in binding sites of from 18,000 to 150,000 per cell. Thus, efficient transduction by an amphotropic retrovirus vector requires high-level expression of the retrovirus receptor Pit2. These results provide the rationale for further examination of the role of receptor levels in inefficient transduction, especially with regard to target cells for gene therapy, where a high transduction rate is often crucial.

FIG. 1

Efficiency of 208F cell transduction following exposure to the LAPSN(PA317) vector. 208F cells were seeded at 5 × 10⁴ cells per 6-cm dish and exposed to LAPSN(PA317) vector beginning the day after seeding. Flow cytometric analysis of alkaline phosphatase expression was performed 2 days after the last exposure to vector. (A) Percentage transduction after a single exposure to various amounts of vector. (B) Percentage transduction after one to six daily exposures to a constant volume of 1 ml of vector. Values represent means ± standard deviations of three independent experiments.

FIG. 2

The effect of rat Pit2 overexpression on transduction by various pseudotypes of the LAPSN vector. Cells were transduced by a single exposure of cells plated the day before at 5 × 10⁴ per 6-cm dish to LAPSN vector at an MOI of 4 for amphotropic and ecotropic pseudotypes and at an MOI of 18 for the GALV pseudotype. The results are means ± standard deviations of three independent experiments for the parental 208F cells, two clones of 208F/LPit2SN cells, and three clones of 208F/LXSN cells.

FIG. 3

Acrylamide gel (6%) analysis of the ¹²⁵I-labeled ASU-hFc fusion protein. Lane 1, protein incubated with β-mercaptoethanol at 100°C for 5 min; lane 2, untreated.

FIG. 4

Flow cytometric analysis of ASU-hFc fusion protein binding to 208F, 208F/LXSN, and 208F/LPit2SN cells. Cells were incubated with ASU-hFc at 4°C for 1 h, washed, incubated with FITC-conjugated rabbit anti-human Fc antibody for 30 min at 4°C, washed, and analyzed by flow cytometry. As a control for nonspecific binding, the fusion protein was replaced with an irrelevant polyclonal human IgG antibody for analysis of 208F cells (IgG isotype control). Mean fluorescence units were calculated from two independent experiments: IgG isotype (dotted line), 5.4 ± 2.1 U; 208F/LXSN (thin line [208F was identical {data not shown}]), 13.2 ± 3.8 U; and 208F/LPit2SN (heavy line), 74 ± 12 U.

FIG. 5

Binding of ¹²⁵I-labeled ASU-hFc fusion protein to 208F/LPit2SN cells. Cell-associated (bound) radioactivity was measured after incubation of 10⁵ cells with labeled fusion protein in a volume of 1 ml at room temperature. (A) Binding as a function of time for different fusion protein concentrations. (B) Binding after a 120-min incubation as a function of fusion protein concentration. The experiment was repeated three times with similar results.

FIG. 6

Scatchard analysis of ASU-hFc binding to 208F, 208F/LXSN, and 208F/LPit2SN cells. The bound/free ratio is plotted against the amount of bound fusion protein, where the bound protein is expressed in molecules per cell and the free protein is expressed in picomolar units. Calculation of the x intercept allows enumeration of binding sites, assuming a binding pattern of one fusion protein homodimer per binding site. Data from a representative experiment are shown.