Stable gene transfer of CCR5 and CXCR4 siRNAs by sleeping beauty transposon system to confer HIV-1 resistance

Abstract

Background: Thus far gene therapy strategies for HIV/AIDS have used either conventional retroviral vectors or lentiviral vectors for gene transfer. Although highly efficient, their use poses a certain degree of risk in terms of viral mediated oncogenesis. Sleeping Beauty (SB) transposon system offers a non-viral method of gene transfer to avoid this possible risk. With respect to conferring HIV resistance, stable knock down of HIV-1 coreceptors CCR5 and CXCR4 by the use of lentiviral vector delivered siRNAs has proved to be a promising strategy to protect cells from HIV-1 infection. In the current studies our aim is to evaluate the utility of SB system for stable gene transfer of CCR5 and CXCR4 siRNA genes to derive HIV resistant cells as a first step towards using this system for gene therapy.

Results: Two well characterized siRNAs against the HIV-1 coreceptors CCR5 and CXCR4 were chosen based on their previous efficacy for the SB transposon gene delivery. The siRNA transgenes were incorporated individually into a modified SB transfer plasmid containing a FACS sortable red fluorescence protein (RFP) reporter and a drug selectable neomycin resistance gene. Gene transfer was achieved by co-delivery with a construct expressing a hyperactive transposase (HSB5) into the GHOST-R3/X4/R5 cell line, which expresses the major HIV receptor CD4 and and the co-receptors CCR5 and CXCR4. SB constructs expressing CCR5 or CXCR4 siRNAs were also transfected into MAGI-CCR5 or MAGI-CXCR4 cell lines, respectively. Near complete downregulation of CCR5 and CXCR4 surface expression was observed in transfected cells. During viral challenge with X4-tropic (NL4.3) or R5-tropic (BaL) HIV-1 strains, the respective transposed cells showed marked viral resistance.

Conclusion: SB transposon system can be used to deliver siRNA genes for stable gene transfer. The siRNA genes against HIV-1 coreceptors CCR5 and CXCR4 are able to downregulate the respective cell surface proteins and thus confer resistance against viral infection by restricting viral entry. These studies have demonstrated for the first time the utility of the non-viral SB system in conferring stable resistance against HIV infection and paved the way for the use of this system for HIV gene therapy studies.

Figure 1

Schematic representation of siRNA SB constructs. A) Control SB transposon plasmid construct with Neo resistance and RFP reporter genes. RFP is driven by a CMV promoter whereas the Neo resistance is expressed via IRES. B) SB transposon construct incorporating anti-CXCR4 siRNA driven by Pol III U6 promoter. C) SB transposon construct incorporating anti-CCR5 siRNA driven by Pol III H1 promoter. D) Plasmid construct encoding the hyperactive transposase under CMV promoter.

Figure 2

Cell surface down regulation of CCR5 or CXCR4 coreceptors in siRNA transfected GHOST-R3/X4/R5 cells. GHOST-R3/X4/R5 cells that constitutively express CCR5 and CXCR4 coreceptors were transfected with control RFP, CCR5 or CXCR4 siRNA constructs. RFP expressing transgenic cells were FACS sorted and cultured. To determine the down regulation of respective coreceptors, the cells were stained with respective FITC tagged antibodies and FACS analyzed. The down regulation of CCR5 coreceptor (Panel A) was determined by comparing CCR5 levels in untransfected (A1), control RFP transfected (A2) and CCR5 siRNA transfected (A3) cells. The CXCR4 coreceptor down regulation is shown by comparing CXCR4 levels in untransfected (B1), control RFP transfected (B2) and CXCR4 siRNA transfected (B3) cells. The percent down regulation of CCR5 (A4) or CXCR4 (B4) coreceptors is also indicated.

Figure 3

HIV-1 challenge of siRNA transposed GHOST-R3/X4/R5 cells. To determine viral resistance, siRNA transposed transgenic cells were challenged with HIV-1 NL4.3 (CXCR4 tropic virus), HIV-1 BaL (CCR5 tropic virus) or HIV-1 89.6 (dual tropic virus) viruses at an MOI of 0.01. On various days post-infection, cell culture supernatants were collected and analyzed for p24 antigen levels by ELISA to determine the levels of viral inhibition. Untransposed (◆), control RFP transposed (■), CXCR4 siRNA transposed (×) or CCR5 siRNA transposed (○). Panel A – NL4.3, Panel B – BaL, Panel C – 89.6.