Background:  An urgent need exists for an HIV-1 microbicide. We hypothesized that RNA interference (RNAi) could be specifically targeted to the cervicovaginal mucosa in vivo and thereby silence genes involved in HIV-1 transmission. RNAi refers to the sequence-specific degradation of RNA following the cellular introduction of short-interfering RNA (siRNA).

Methods:  We first optimized siRNA delivery to vaginal tissue in vivo using C57/BL6 mice. These investigations revealed that liposomal preparations were the most effective delivery vehicles. We next characterized siRNA tissue distribution in vivo by vaginally administering fluorescent siRNA/liposomes.

To determine the kinetics of siRNA activity, we chose the nuclear membrane protein lamin A/C as a target given its constitutive expression in all somatic tissues of the adult mouse. We introduced lamin A/C siRNA/liposomes to groups of 10 animals and quantified corresponding mRNA and protein levels by real time polymerase chain reaction (PCR) and Western blot (WB), respectively, over a 10-day period. Untreated animals and those treated in the same manner with an irrelevant siRNA or liposome-only solution served as relevant controls.

Results:  Liposomal siRNA complexes penetrated the entire thickness of the murine vagina as demonstrated by confocal microscopy. Within individual cells, siRNA characteristically localized to peri-nuclear areas. A single vaginal administration of lamin A/C siRNA reduced corresponding mRNA levels by 86% and 84% on days 2 and 4, respectively, compared to control animals. At day 7, we observed slight diminution of gene silencing with mRNA levels reduced by 79%. By day 10, however, lamin A/C mRNA had returned to pretreatment levels. Thus, significant gene silencing persisted over a 7-day period, as analyzed by one-way analysis of variance with significance testing by Bodferroni-adjusted t-tests. These findings were confirmed by WB for lamin A/C vaginal expression. Liposomal siRNA formulations proved nontoxic by microscopic analysis of tissue sections.

Conclusions:  Direct mucosal application of liposome-complexed siRNA leads to gene-specific silencing in cervico-vaginal epithelia. RNAi may be exploited to knock down the vaginal expression of gene products such as CCR5 and thereby may emerge as a novel HIV-1microbicide.

Keywords: siRNA; microbicides; gene therapy