Helper-free foamy virus vectors

Abstract

Retroviral vectors based on human foamy virus (HFV) have been developed and show promise as gene therapy vehicles. Here we describe a method for the production of HFV vector stocks free of detectable helper virus. The helper and vector plasmid constructs used both lack the HFV bel genes, so recombination between these constructs cannot create a wild-type virus. A fusion promoter that combines portions of the cytomegalovirus (CMV) immediate-early and HFV long terminal repeat (LTR) promoters was used to drive expression of both the helper and vector constructs. The CMV-LTR fusion promoter allows for HFV vector production in the absence of the Bel-1 trans-activator protein, which would otherwise be necessary for efficient transcription from the HFV LTR. Vector stocks containing either neomycin phosphotransferase or alkaline phosphatase reporter genes were produced by transient transfection at titers greater than 10(5) transducing units/ml. G418-resistant BHK-21 cells obtained by transduction with neo vectors contained randomly integrated HFV vector proviruses without detectable deletions or rearrangements. The vector stocks generated were free of replication-competent retrovirus (RCR), as determined by assays for LTR trans-activation and a marker rescue assay developed here for the detection of Bel-independent RCR.

FIG. 1

Bel-independent HFV vector constructs. (A) Map of the wt HFV provirus pHSRV13, indicating sites of transcriptional transactivation by the Bel1 protein. The positions of the LTRs and of the gag, pol, env, and bel genes are indicated. (B) Maps of the Bel-independent vector constructs engineered with fusion promoters consisting of the CMV promoter in place of the HFV LTR 5′ U3 region. Transcription is initiated at the R region of the LTR, independently of Bel1. The engineered XbaI site used to ligate the CMV and HFV promoter elements is underlined and the reported HFV start site is indicated by an asterisk above the pCGPMAPΔBel construct. The positions of the CMV fusion promoter, MLV LTR promoter (M), alkaline phosphatase gene (AP), SV40 early promoter (S), and neo gene are indicated. (C) Map of the pCGPES helper construct, showing the locations of the CMV–HFV LTR fusion promoter (sequence slightly different than that of pCGPMAPΔBel), HFV gag, pol, and env genes, and the SV40 polyadenylation site (SpA). (D) Structure of integrated vector provirus resulting from transduction of Bel-independent vectors. The wt HFV 5′ LTR is regenerated by reverse transcription prior to integration. The locations of relevant restriction sites and the probe used for Southern analysis are shown. Transcription start sites are indicated by arrows.

FIG. 2

Vector production in various cell lines. The helper plasmid pCGPES and vector plasmid pCGPMAPΔBel were cotransfected into the indicated cell lines and cell-free supernatants were harvested 72 hr later, filtered, and titered on FAB cells. Values shown are titers (AP FFU/ml) with means and standard errors from three independent measurements for each cell line. In some cases, standard errors were too small to be displayed in the figure. Controls of untransfected cells and cells transfected with vector plasmid pCGPMAPΔBel alone produced no AP transducing units (<1 AP FFU/ml). Helper virus contamination was not detected by FAB assay (<1 BFFU/ml).

FIG. 3

Time course of vector production by transient transfection of 293T cells. Three independent transfections were performed with plasmids pCGPES and pCGPMAPΔBel, and at 24, 36, 48, 56, 72, and 120 hr 100 μ l of vector-containing medium was removed from each well and frozen at −80°C until assayed. Samples were then thawed together at room temperature and titered on FAB cells. Controls of cells transfected with vector plasmid alone were negative (<1 AP FFU/ml). Values shown are titers (AP FFU/ml), with each symbol type representing a different transfection.

FIG. 4

Comparison of vector harvest methods. 293T cells were transfected with both the helper construct pCGPES and vector construct pCGPMAPΔBel, and 72 hr later supernatants (vector-containing medium) or suspensions of cells and supernatants were harvested as follows and titered on FAB cells. Harvest conditions included supernatant filtered without freezing (SUP); filtered supernatant that was frozen at −80°C for 30 min, then thawed at room temperature (SUP 1×F/T); filtered supernatant with glycerol added to 40% (v/v) that was frozen at −80°C for 30 min, then thawed at room temperature (SUP 1×F/T GLYC); supernatant combined with a suspension of the transfected cells that was frozen at −80°C for 30 min, thawed at room temperature, then filtered (SUSP 1×F/T); or supernatant combined with a suspension of the transfected cells that was frozen in an ethanol–dry ice bath and thawed at 37°C three times, then filtered (SUSP 3×F/T). Controls of untransfected cells or cells transfected with vector plasmid pCGPMAPΔBel alone produced no AP FFU (<1/ml). Supernatant from a control well transfected with the same transfection mixture used for all treatments was tested by FAB assay and did not contain Bel-dependent RCR (<1 BFFU/ml). Mean titer values (AP FFU/ml) with standard errors from three independent measurements are plotted. Values in parentheses indicate the fold reduction as compared with unfrozen supernatants.

FIG. 5

Foamy marker rescue assay. An FMR cell is depicted on the left with its integrated, env-deficient neo vector provirus. On the right an FMR cell is shown with both RCR and vector proviruses after infection by an env⁺ RCR virion. This cell produces additional RCR particles that can spread through the culture, and neo vector particles that can transduce BHK-21 cells. The positions of gag, pol, env, and neo genes in the proviruses are indicated. The CMV-HFV LTR fusion promoter (C|F), the HFV LTR (F), and a novel Bel-independent LTR (X) are indicated.

FIG. 6

Southern analysis of integrated Bel-independent vector genomes. High molecular weight DNAs isolated from 10 independent G418-resistant clones of BHK-21 cells transduced by pCGPSNΔBel (lanes 1–10) or untransduced BHK-21 cells were digested with the indicated enzymes and probed for neo sequences. Samples were digested with either EcoNI, which produces a 7.7-kb fragment from an intact provirus (A) or with EcoRI, which cuts once within the vector (B). Uneven loading decreased the vector signal in lanes 1 and 3 (A) and lane 10 (B). See Fig. 1 for restriction sites and probe map.