A bovine papillomavirus-1 based vector restores the function of the low-density lipoprotein receptor in the receptor-deficient CHO-ldlA7 cell line

Abstract

Background: The rationale of using bovine papillomavirus-1 (BPV-1) derived vectors in gene therapy protocols lies in their episomal maintenance at intermediate to high copy number, and stable, high-level expression of the gene products. We constructed the BPV-1 based vector harbouring the human low-density lipoprotein receptor (LDLR) gene cDNA and tested its ability to restore the function of the LDLR in the receptor-deficient cell line CHO-ldlA7.

Results: The introduced vector p3.7LDL produced functionally active LDL receptors in the receptor-deficient cell line CHO-ldlA7 during the 32-week period of observation as determined by the internalisation assay with the labelled LDL particles.

Conclusion: Bovine papillomavirus type-1 (BPV-1)-derived vectors could be suitable for gene therapy due to their episomal maintenance at intermediate to high copy number and stable, high-level expression of the gene products. The constructed BPV-1 based vector p3.7LDL produced functionally active LDL receptors in the LDLR-deficient cell line CHO-ldlA7 during the 32-week period of observation. In vivo experiments should reveal, whether 1-5% transfection efficiency obtained in the current work is sufficient to bring about detectable and clinically significant lowering of the amount of circulating LDL cholesterol particles.

Figure 1

p3.7LDL – a bovine papillomavirus-1 (BPV-1) derived vector harbouring the 2.6 kb hLDLR cDNA fragment.

Figure 2

Transient replicative properties of the expression vectors. CHO cells were electroporated with 1, 2 and 5 μg of p3.7LDL (lanes 2,3,4) and the empty self-replicating vector TK3.7 (lanes 6, 7 8). Low-molecular weight DNA was digested with DpnI and the linearising enzyme Bpu1102I. Fragments were analysed by Southern blotting. The corresponding marker DNAs (200 pg each) were digested with Bpu1102I (lanes 1 and 5). Hybridisation probe was a 1 kb BPV-1 origin fragment. Exposure 12 hrs.

Figure 3

The Southern blot analysis of native and the p3.7LDL-transfected CHO-ldlA7 cell clones. Lanes 1 to 5 – Bpu1102 digested genomic DNA from different transfected cell clones; lane 6-Bpu1102 digested genomic DNA from native CHO-ldlA7 cells; lane 7-positive control-the Bpu 1102 linearised p3.7LDL, which gives rise to a 19.3 kb band. The same linearised vector was used as a hybridisation probe. Multiple bands in transfected clones suggest integration of the vector sequences into the host cell genome.

Figure 5

RT-PCR analysis of the native and the p3.7LDL-transfected CHO-ldlA7 cell clones. Lanes 1 to 5 – RT-PCR products from different transfected cell clones; lane 6 – native CHO-ldlA7 cells; lane 7 – negative control for cDNA synthesis (without RNA); lane 8 – negative control for PCR (reaction without cDNA); lane 9–100 bp DNA ladder (Fermentas).

Figure 4

Figure 4a. Fluorescence in situ analysis of the p3.7LDL-transfected CHO-ldlA7 cells. The probe, generated from the p3.7LDL gives cross-hybridisation signals represented by yellow double dots on chromosomes stained with propidium iodide. This can be explained by integration of the vector sequences into the host cell genome The discrete yellow dots dispersed all over the chromosomes also correspond to plasmid-specific signals and suggest the presence of an episomal vector. Figure 4b. Fluorescence in situ analysis of the native CHO-ldlA7 cells (negative control).

Figure 6

Immunofluorescence-based detection of the LDLR protein in the p3.7LDL-transfected and the native CHO-ldlA7 cells. Supernatant of IgG2b monoclonal antibody (CRL-1691, ATCC) against hLDLR was used as the primary and anti-sheep FITC conjugated as the secondary antibody. Samples were treated with SlowFade™ Light Antifade reagent and viewed under a fluorescence microscope. In transfected cells, the fluorescent signal is detectable in the cytoplasm generally and in the Golgi complex particularly. A weak background signal is visible in the native cell population

Figure 7

Assessment of the LDL receptor functionality by the internalisation assay. The p3.7LDL-transfected (A) and the native (B) CHOldlA7 cells were incubated with DiI-LDL particles and after fixation viewed by fluorescence microscopy. Signal from the particles (seen as red spots) is visible inside the transfected cells. Some non-transfected cells have labelled particles associated with cell membranes.