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

Tamhane M, Akkina R - AIDS Res Ther (2008)

Bottom Line: During viral challenge with X4-tropic (NL4.3) or R5-tropic (BaL) HIV-1 strains, the respective transposed cells showed marked viral resistance.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Dept, Microbiology, Immunology and Pathology, Colorado State University, Fort Collins, Colorado 80523, USA.

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.

No MeSH data available.


Related in: MedlinePlus

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.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2533343&req=5

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.

Mentions: The above data showed that SB transposed siRNAs are stably integrated into respective cells. We next evaluated if the stably gene modified cells show the effect of siRNA mediated gene silencing. Accordingly, the transposed cells were analysed for CXCR4 or CCR5 surface expression by FACS (Figure 2). Our results showed about 94% down-regulation of CXCR4 expression and a 97% down-regulation of CCR5 in GHOST-R3/X4/R5 cells transposed with CXCR4 or CCR5 siRNAs respectively. In the MAGI-CXCR4 cell line, the CXCR4 expression was reduced by 98% by the respective siRNA, while MAGI-CCR5 cells showed a 99% reduction in CCR5 levels as a result of respective transposon mediated siRNA expression (data not shown). Cells transposed with control SB construct without siRNA insert showed no decrease in coreceptor expression with levels similar to that shown by control unmanipulated cells. The levels of coreceptor down regulation obtained with these siRNAs in SB system are similar to that seen with that delivered via lentiviral vectors (data not shown). These results confirmed the efficacy of the respective siRNAs in mediating gene silencing of the HIV-1 coreceptors.


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

Tamhane M, Akkina R - AIDS Res Ther (2008)

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.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2533343&req=5

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.
Mentions: The above data showed that SB transposed siRNAs are stably integrated into respective cells. We next evaluated if the stably gene modified cells show the effect of siRNA mediated gene silencing. Accordingly, the transposed cells were analysed for CXCR4 or CCR5 surface expression by FACS (Figure 2). Our results showed about 94% down-regulation of CXCR4 expression and a 97% down-regulation of CCR5 in GHOST-R3/X4/R5 cells transposed with CXCR4 or CCR5 siRNAs respectively. In the MAGI-CXCR4 cell line, the CXCR4 expression was reduced by 98% by the respective siRNA, while MAGI-CCR5 cells showed a 99% reduction in CCR5 levels as a result of respective transposon mediated siRNA expression (data not shown). Cells transposed with control SB construct without siRNA insert showed no decrease in coreceptor expression with levels similar to that shown by control unmanipulated cells. The levels of coreceptor down regulation obtained with these siRNAs in SB system are similar to that seen with that delivered via lentiviral vectors (data not shown). These results confirmed the efficacy of the respective siRNAs in mediating gene silencing of the HIV-1 coreceptors.

Bottom Line: During viral challenge with X4-tropic (NL4.3) or R5-tropic (BaL) HIV-1 strains, the respective transposed cells showed marked viral resistance.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Dept, Microbiology, Immunology and Pathology, Colorado State University, Fort Collins, Colorado 80523, USA.

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.

No MeSH data available.


Related in: MedlinePlus