Inhibition of HIV transmission in human cervicovaginal explants and humanized mice using CD4 aptamer-siRNA chimeras
Lee Adam Wheeler, Radiana Trifonova, Vladimir Vrbanac, Emre Basar, Shannon McKernan, Zhan Xu, Edward Seung, Maud Deruaz, Tim Dudek, Jon Ivar Einarsson, Linda Yang, Todd M. Allen, Andrew D. Luster, Andrew M. Tager, Derek M. Dykxhoorn, Judy Lieberman
Lee Adam Wheeler, Radiana Trifonova, Vladimir Vrbanac, Emre Basar, Shannon McKernan, Zhan Xu, Edward Seung, Maud Deruaz, Tim Dudek, Jon Ivar Einarsson, Linda Yang, Todd M. Allen, Andrew D. Luster, Andrew M. Tager, Derek M. Dykxhoorn, Judy Lieberman
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Research Article AIDS/HIV

Inhibition of HIV transmission in human cervicovaginal explants and humanized mice using CD4 aptamer-siRNA chimeras

Abstract

The continued spread of the HIV epidemic underscores the need to interrupt transmission. One attractive strategy is a topical vaginal microbicide. Sexual transmission of herpes simplex virus type 2 (HSV-2) in mice can be inhibited by intravaginal siRNA application. To overcome the challenges of knocking down gene expression in immune cells susceptible to HIV infection, we used chimeric RNAs composed of an aptamer fused to an siRNA for targeted gene knockdown in cells bearing an aptamer-binding receptor. Here, we showed that CD4 aptamer-siRNA chimeras (CD4-AsiCs) specifically suppress gene expression in CD4+ T cells and macrophages in vitro, in polarized cervicovaginal tissue explants, and in the female genital tract of humanized mice. CD4-AsiCs do not activate lymphocytes or stimulate innate immunity. CD4-AsiCs that knock down HIV genes and/or CCR5 inhibited HIV infection in vitro and in tissue explants. When applied intravaginally to humanized mice, CD4-AsiCs protected against HIV vaginal transmission. Thus, CD4-AsiCs could be used as the active ingredient of a microbicide to prevent HIV sexual transmission.

Authors

Lee Adam Wheeler, Radiana Trifonova, Vladimir Vrbanac, Emre Basar, Shannon McKernan, Zhan Xu, Edward Seung, Maud Deruaz, Tim Dudek, Jon Ivar Einarsson, Linda Yang, Todd M. Allen, Andrew D. Luster, Andrew M. Tager, Derek M. Dykxhoorn, Judy Lieberman

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Figure 2

CD4-AsiCs inhibit HIV replication in vitro.

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CD4-AsiCs inhibit HIV replication in vitro. MDMs (A–C) and CD4+ T cells ...
MDMs (A–C) and CD4+ T cells (D and E) were infected for 48 hours with HIV-1BaL and HIV-1IIIb, respectively, and then treated with CD4-AsiCs or PSMA-AsiCs bearing gag and vif (g/v) siRNAs. Scrambled siRNA chimeras and CD4 aptamers were controls. Transfection controls used OF (A and B) or nucleofection (D and E). (A and D) Intracellular p24 expression, relative to infected mock-treated cultures, was measured by flow cytometry 48 hours later. CD4-AsiCs inhibited HIV replication (mean ± SEM normalized to mock; n = 3; *P < 0.05, **P < 0.005, 2-tailed t test). Insets show representative histograms of MDMs and CD4+ T lymphocytes treated with gag and vif CD4-AsiCs (gray, uninfected; blue, infected mock-treated; red, infected CD4-AsiC–treated). (C) HIV infection was also evaluated by fluorescence in situ hybridization (original magnification, ×60) using FITC-labeled probes complementary to HIV genomic RNA. HIV RNA was virtually undetectable in MDMs treated with 4 μM total final concentration of CD4-AsiCs. (B and E) To evaluate gene silencing independently of the effect of the CD4 aptamer on blocking viral entry, HIV replication was assessed by infection with VSV(G)-pseudotyped virus containing a luciferase reporter gene. Primary MDMs and CD4+ T cells were pretreated for 48 hours before infection with 4-μM mixtures of CD4- or PSMA-AsiCs targeting gag and vif or containing scrambled siRNAs. Luciferase activity, measured 48 hours later, was significantly inhibited in cells treated with CD4-AsiCs directed against either viral or luciferase genes (mean ± SEM normalized to mock; n = 3; *P < 0.05, **P < 0.005).