Background:  The HIV-1 co-receptor CCR5 has been thought to be a relevant target for small interfering RNA (siRNA)-based therapeutics. A number of siRNA have been shown to have antiviral activity in cell culture assays and in vivo models. However, recent findings suggest that siRNA can stimulate innate cytokine responses in mammals.

Methods:  We evaluated the effect of siRNA targeting the HIV co-receptor CCR5 previously shown to block HIV replication. Inhibition of virus production was tested in U87^­CD4⁺/CCR5⁺ cells. We studied whether these siRNA induce production of cytokines, by cytokine bead array assays, and how they could affect HIV replication.

Results:  All siRNA agents down-regulated the expression of CCR5, albeit with different efficiency (51 to 74% down-regulation), blocked HIV-induced syncytium formation between HIV-1 BaL-infected and uninfected CD4⁺ cells or blocked single-round HIV-1 infection as measured by a luciferase reporter assay (46 to 83% inhibition). siRNA directed against CCR5 did not affect replication of a vesicular stomatotis pseudotyped virus, suggesting that inhibition of HIV replication was specific of CCR5 down-regulation. However, 2 of 4 siRNA tested induced the production of inflammatory cytokines interleukin (IL)-6 (6-fold induction) and IL-8 (9-fold induction) but no interferon (IFN)-a, IFN-g, tumor necrosis factor (TNF)-a, macrophage chemotactic protein (MCP)-1, macrophage inhibitory protein (MIP)-1a, MIP-1b, RANTES, interleukin (IL)-1b, IL-10, or IL-12p70 cytokine induction was noted. Although IL-6, IL-8, or both cytokines did not affect the anti-HIV activities of siRNA in U87^­CD4⁺/CCR5⁺ cells, we found that, upon mitogenic stimulation, CD4⁺ cells from HIV-infected patients (n = 7) showed increased levels of IL-6 compared to non-infected individuals (n = 5).

Conclusions:  Secreted IFN-a has been considered as a reporter of non-specific effects induced by double-stranded RNA and siRNA. However, in the absence of detectable IFN-a, IL-6, or IL-8 may represent markers of non-specific effect triggered by siRNA. Furthermore, gene specific (i.e. CCR5) siRNA may have a differential induction of non-specific effects that may increase rather than block HIV replication. Selection of siRNA sequences devoid of unwanted immunostimulatory activity should represent an important element for successful siRNA antiviral therapy.