Durable knockdown and protection from HIV transmission in humanized mice treated with gel-formulated CD4 aptamer-siRNA chimeras.

The continued spread of HIV underscores the need to interrupt transmission. One attractive strategy, in the absence of an effective vaccine, is a topical microbicide, but the need for application around the time of sexual intercourse leads to poor patient compliance. Intravaginal (IVAG) application of CD4 aptamer-siRNA chimeras (CD4-AsiCs) targeting the HIV coreceptor CCR5, gag, and vif protected humanized mice from sexual transmission. In non-dividing cells and tissue, RNAi-mediated gene knockdown lasts for several weeks, providing an opportunity for infrequent dosing not temporally linked to sexual intercourse, when compliance is challenging. Here, we investigate the durability of gene knockdown and viral inhibition, protection afforded by CCR5 or HIV gene knockdown on their own, and effectiveness of CD4-AsiCs formulated in a gel in polarized human cervicovaginal explants and in humanized mice. CD4-AsiC-mediated gene knockdown persisted for several weeks. Cell-specific gene knockdown and protection were comparable in a hydroxyethylcellulose gel formulation. CD4-AsiCs against CCR5 or gag/vif performed as well as a cocktail in humanized mice. Transmission was completely blocked by CCR5 CD4-AsiCs applied 2 days before challenge. Significant, but incomplete, protection also occurred when exposure was delayed for 4 or 6 days. CD4-AsiCs targeting gag/vif provided some protection when administered only after exposure. These data suggest that CD4-AsiCs are a promising approach for developing an HIV microbicide.

HIV DNA is heavily uracilated, which protects it from autointegration.

Human immune cells infected by HIV naturally contain high uracil content, and HIV reverse transcriptase (RT) does not distinguish between dUTP and dTTP. Many DNA viruses and retroviruses encode a dUTPase or uracil-DNA glycosylase (UNG) to counteract uracil incorporation. However, although HIV virions are thought to contain cellular UNG2, replication of HIV produced in cells lacking UNG activity does not appear to be impaired. Here we show that HIV reverse transcripts generated in primary human immune cells are heavily uracilated (>500 uracils per 10 kb HIV genome). We find that HIV DNA uracilation, rather than being dangerous, may promote the early phase of the viral life cycle. Shortly after reverse transcription, the ends of the HIV DNA are activated by the viral integrase (IN) in preparation for chromosomal insertion. However, the activated ends can attack the viral DNA itself in a suicidal side pathway, called autointegration. We find here that uracilation of target DNA inhibits the strand transfer of HIV DNA ends by IN, thereby inhibiting autointegration and facilitating chromosomal integration and viral replication. When uracilation is increased by incubating uracil-poor cells in the presence of increasing concentrations of dUTP or by infecting with virus that contains the cytosine deaminase APOBEC3G (A3G), the proportion of reverse transcripts that undergo suicidal autointegration decreases. Thus, HIV tolerates, or even benefits from, nonmutagenic uracil incorporation during reverse transcription in human immune cells.

Silencing sexually transmitted infections: topical siRNA-based interventions for the prevention of HIV and HSV.

The global impact of sexually transmitted infections (STIs) is significant. The sexual transmission of viruses such as herpes simplex virus type-2 (HSV-2) and the human immunodeficiency virus type-1 (HIV-1), has been especially difficult to control. To date, no effective vaccines have been developed to prevent the transmission of these STIs. Although antiretroviral drugs have been remarkably successful in treating the symptoms associated with these viral infections, the feasibility of their widespread use for prevention purposes may be more limited. Microbicides might provide an attractive alternative option to reduce their spread. In particular, topically applied small inhibitory RNAs (siRNAs) have been shown to not only block transmission of viral STIs to mucosal tissues both in vitro and in vivo, but also confer durable knockdown of target gene expression, thereby circumventing the need to apply a microbicide around the time of sexual encounter, when compliance is mostly difficult. Despite numerous clinical trials currently testing the efficacy of siRNA-based therapeutics, they have yet to be approved for use in the treatment of viral STIs. While several obstacles to their successful implementation in the clinic still exist, promising preclinical studies suggest that siRNAs are a viable modality for the future prevention and treatment of HSV and HIV.

Tension pneumocephalus mimicking septic shock: a case report.

Tension pneumocephalus can lead to rapid neurologic deterioration. We report for the first time its association with aseptic systemic inflammatory response syndrome mimicking septic shock and the efficacy of prompt neurosurgical intervention and critical care support in treating this condition. A 64-year-old man underwent 2-stage olfactory groove meningioma resection. The patient developed altered mental status and gait instability on postoperative day 6. Imaging showed significant pneumocephalus. The patient subsequently developed worsening mental status, respiratory failure, and profound shock requiring multiple vasopressors. Bedside needle decompression, identification and repair of the cranial fossa defect, and critical care support led to improved mental status and reversal of shock and multiorgan dysfunction. Thorough evaluation revealed no evidence of an underlying infection. In this case, tension pneumocephalus incited an aseptic systemic inflammatory response syndrome mimicking septic shock. Prompt neurosurgical correction of pneumocephalus and critical care support not only improved neurologic status, but also reversed shock. Such a complication indicates the importance of close monitoring of patients with progressive pneumocephalus.

TREX1 Knockdown Induces an Interferon Response to HIV that Delays Viral Infection in Humanized Mice.

Despite their antiviral effect, the in vivo effect of interferons on HIV transmission is difficult to predict, because interferons also activate and recruit HIV-susceptible cells to sites of infection. HIV does not normally induce type I interferons in infected cells, but does if TREX1 is knocked down. Here, we investigated the effect of topical TREX1 knockdown and local interferon production on HIV transmission in human cervicovaginal explants and humanized mice. In explants in which TREX1 was knocked down, HIV induced interferons, which blocked infection. In humanized mice, even though TREX1 knockdown increased infiltrating immune cells, it delayed viral replication for 3-4 weeks. Similarly intravaginal application of type I interferons the day before HIV infection induced interferon responsive genes, reduced inflammation, and decreased viral replication. However, intravenous interferon enhanced inflammation and infection. Thus, in models of human sexual transmission, a localized interferon response inhibits HIV transmission but systemic interferons do not.

Gene Knockdown by EpCAM Aptamer-siRNA Chimeras Suppresses Epithelial Breast Cancers and Their Tumor-Initiating Cells.

Effective therapeutic strategies for in vivo siRNA delivery to knockdown genes in cells outside the liver are needed to harness RNA interference for treating cancer. EpCAM is a tumor-associated antigen highly expressed on common epithelial cancers and their tumor-initiating cells (TIC, also known as cancer stem cells). Here, we show that aptamer-siRNA chimeras (AsiC, an EpCAM aptamer linked to an siRNA sense strand and annealed to the siRNA antisense strand) are selectively taken up and knock down gene expression in EpCAM(+) cancer cells in vitro and in human cancer biopsy tissues. PLK1 EpCAM-AsiCs inhibit colony and mammosphere formation (in vitro TIC assays) and tumor initiation by EpCAM(+) luminal and basal-A triple-negative breast cancer (TNBC) cell lines, but not EpCAM(-) mesenchymal basal-B TNBCs, in nude mice. Subcutaneously administered EpCAM-AsiCs concentrate in EpCAM(+) Her2(+) and TNBC tumors and suppress their growth. Thus, EpCAM-AsiCs provide an attractive approach for treating epithelial cancer.

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

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.