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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

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.
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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.

Mentions: To determine if down regulation of CCR5 and CXCR4 coreceptors conferred viral resistance, siRNA transgenic GHOST-R3/X4/R5 cells were challenged with X4-tropic (NL4-3), R5-tropic (BaL-1) and dual coreceptor tropic HIV-1 89.6 strain. Antigen ELISAs to detect viral p24 in culture supernatants were performed on various days post-infection up to three weeks (Figure 3). When challenged with X4-tropic HIV-1 NL4.3, GHOST-R3/X4/R5 cells expressing CXCR4 siRNA showed a 10 fold decrease in virus production as compared to control non-transgenic cells on day 10 post-infection. The level of viral inhibition reached upto 14 fold through day 21 post-infection. In contrast CCR5 siRNA expressing GHOST-R3/X4/R5 cells failed to show any inhibition of virus production against X4 tropic HIV-1 NL4.3. Viral challenge of GHOST-R3/X4/R5 cells expressing CCR5 siRNA with the R5-tropic HIV-1 BaL resulted in an 8 fold reduction in virus production on day 10 post-infection, which doubled to 16 fold on days 14 and 21 post-infection. GHOST-R3/X4/R5 cells expressing CXCR4 siRNA served as a negative control as they showed similar levels of infection seen in control non-transgenic cells with the R5-tropic virus challenge. In dual-tropic HIV-1 89.6 viral challenges, neither of the individual CXCR4 siRNA or CCR5 siRNA expressing GHOST-R3/X4/R5 cells showed significant protection as expected since the challenge virus could use either of the coreceptors. However there was a moderate decrease in the virus production on day 21 as compared to unmanipulated cells. Cells transposed with SB control construct without anti-HIV transgenes showed similar levels of infection as the unmanipulated cells for all three HIV-1 strains. We also challenged SB transposed MAGI-CCR5 and MAGI-CXCR4 cells with R5 or X4 tropic viral strains respectively and found similar levels of resistance (data not shown). These data collectively showed that the respective SB system delivered siRNAs are functional and mediate viral resistance.


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)

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.
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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.
Mentions: To determine if down regulation of CCR5 and CXCR4 coreceptors conferred viral resistance, siRNA transgenic GHOST-R3/X4/R5 cells were challenged with X4-tropic (NL4-3), R5-tropic (BaL-1) and dual coreceptor tropic HIV-1 89.6 strain. Antigen ELISAs to detect viral p24 in culture supernatants were performed on various days post-infection up to three weeks (Figure 3). When challenged with X4-tropic HIV-1 NL4.3, GHOST-R3/X4/R5 cells expressing CXCR4 siRNA showed a 10 fold decrease in virus production as compared to control non-transgenic cells on day 10 post-infection. The level of viral inhibition reached upto 14 fold through day 21 post-infection. In contrast CCR5 siRNA expressing GHOST-R3/X4/R5 cells failed to show any inhibition of virus production against X4 tropic HIV-1 NL4.3. Viral challenge of GHOST-R3/X4/R5 cells expressing CCR5 siRNA with the R5-tropic HIV-1 BaL resulted in an 8 fold reduction in virus production on day 10 post-infection, which doubled to 16 fold on days 14 and 21 post-infection. GHOST-R3/X4/R5 cells expressing CXCR4 siRNA served as a negative control as they showed similar levels of infection seen in control non-transgenic cells with the R5-tropic virus challenge. In dual-tropic HIV-1 89.6 viral challenges, neither of the individual CXCR4 siRNA or CCR5 siRNA expressing GHOST-R3/X4/R5 cells showed significant protection as expected since the challenge virus could use either of the coreceptors. However there was a moderate decrease in the virus production on day 21 as compared to unmanipulated cells. Cells transposed with SB control construct without anti-HIV transgenes showed similar levels of infection as the unmanipulated cells for all three HIV-1 strains. We also challenged SB transposed MAGI-CCR5 and MAGI-CXCR4 cells with R5 or X4 tropic viral strains respectively and found similar levels of resistance (data not shown). These data collectively showed that the respective SB system delivered siRNAs are functional and mediate viral resistance.

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