Promoter Targeting shRNA Suppresses HIV-1 Infection In vivo Through Transcriptional Gene Silencing

Abstract

Despite prolonged and intensive application, combined antiretroviral therapy cannot eradicate human immunodeficiency virus (HIV)-1 because it is harbored as a latent infection, surviving for long periods of time. Alternative approaches are required to overcome the limitations of current therapy. We have been developing a short interfering RNA (siRNA) gene silencing approach. Certain siRNAs targeting promoter regions of genes induce transcriptional gene silencing. We previously reported substantial transcriptional gene silencing of HIV-1 replication by an siRNA targeting the HIV-1 promoter in vitro. In this study, we show that this siRNA, expressed as a short hairpin RNA (shRNA) (shPromA-JRFL) delivered by lentiviral transduction of human peripheral blood mononuclear cells (PBMCs), which are then used to reconstitute NOJ mice, is able to inhibit HIV-1 replication in vivo, whereas a three-base mismatched variant (shPromA-M2) does not. In shPromA-JRFL–treated mice, HIV-1 RNA in serum is significantly reduced, and the ratio of CD4+/CD8+ T cells is significantly elevated. Expression levels of the antisense RNA strand inversely correlates with HIV-1 RNA in serum. The silenced HIV-1 can be reactivated by T-cell activation in ex vivo cultures. HIV-1 suppression is not due to offtarget effects of shPromA-JRFL. These data provide “proof-of principle” that an shRNA targeting the HIV-1 promoter is able to suppress HIV-1 replication in vivo.

Post-transcriptional gene silencing, transcriptional gene silencing and human immunodeficiency virus

“…In particular, the much awaited identification and characterization of a functional nuclear mammalian RITS complex, because there are apparently no RNAi proteins with homology to Tas3 and Chp1 present in the nucleus and AGO-1 is non-catalytic. At present, most of the evidence of mammalian sncRNA-AGO-1 directed TGS relies on synthetic siRNAs or shRNAs driving TGS to control infectious agents, such as HIV-1 [113] , or cellular genes that support viral replication [114] . Nonetheless, the relatively slow accumulation of evidence has supported the existence of this functional pathway, with evidence for miRNA-induced TGS in senescence [107] and in differentiation [65] .…”

Recent advances in RNAi-based strategies for therapy and prevention of HIV-1/AIDS

“…Suzuki et al transduced human PBMCs with lentivirus encoding shRNAs targeting the promoter region of the HIV LTR (which mediates transcriptional gene silencing by inducing epigenetic changes) and infused these cells into NOD/SCID mice deficient for Janus kinase 3 (NOJ mice). These mice when infected with HIV-1 were resistant to CD4 T cell depletion and had a reduced viral load compared with controls [139]. We have transduced PBMCs from HIV-seropositive individuals with a lentiviral vector expressing seven shRNAs and shown its effectiveness in suppressing endogenous virus activation in hu-PBL mice [107].…”

Achieving HIV-1 Control through RNA-Directed Gene Regulation

“…The HIV-1 promoter region targeted by the siRNA sequence was the tandem repeat of NF-κB binding motifs in the 5′ LTR (Figure 2a), which has also been of interest in other HIV studies due to its potent transcriptional activation of the virus [81,82,83]. We have demonstrated profound silencing of HIV-1 replication up to 1000-fold from a single treatment of siRNA or shRNA delivered by lentivirus vector, both in vitro [54,84,85,86,87] using T cell lines, PBMC and monocyte-derived macrophages, and in vivo using PBMCs [88]. To distinguish a TGS effect from a PTGS effect, we performed nuclear run-on assays, and definitively confirmed the RNA therapeutic suppressed HIV-1 via the TGS pathway [86,87].…”

“…To demonstrate in vivo activity of shPromA we employed an acute model of HIV in the (NOD)/SCID/Janus kinase 3 (NOJ) knockout humanized mouse model [88]. The lentiviral-delivered shPromA was used to transduce human PBMCs, which were transplanted into NOJ mice.…”

“…The lentiviral-delivered shPromA was used to transduce human PBMCs, which were transplanted into NOJ mice. Following HIV-1 challenge with strain JR-FL, mice reconstituted with the anti-HIV shPromA showed significantly lower plasma viral loads and a normal CD4:CD8 T cell ratio, while the control group treated with cells transduced by the inactive mutated version of shProm A, shPromA-M2, showed the expected course of acute HIV-1 infection, with high plasma viral load and low CD4+ T cell numbers resulting in rapid onset of immunodeficiency [88]. The protective effect observed against HIV-1 likely corresponds to the “latent-like” state induced in cell line and primary cell studies, which were induced by epigenetic silencing.…”