225 Sustained siRNA-mediated HIV Inhibition in Primary Macrophages P. Shankar*1, E. Song1, S.K. Lee1, D.M. Dykxhoom2, C. Novina2, K. Crawford1, J. Cerny1, P.A. Sharp2, J. Lieberman1, M.N. Swamy1 1Ctr for Blood Res, Harvard Med Sch, Boston, MA and 2Ctr for Cancer Res, MIT, Cambridge, MA
Background: Our previous study has shown that synthetic 21-23 nucleotide siRNA duplexes suppress HIV replication in cell lines and proliferating CD4 T-cells. However, the silencing effect peaks around 96 h, but tapers off thereafter, presumably because of siRNA dilution with cell division or degradation inside the cells. In this study, we investigated whether siRNAs targeting a viral structural gene p24 or the HIV-1 coreceptor CCR5 could mediate stable suppression of HIV-1 infection in terminally differentiated non-dividing macrophages, which constitute an important reservoir of HIV.
Methods: Macrophages generated from adherent peripheral blood mononuclear cells from normal volunteer donors were transfected with siRNA duplexes targeting CCR5 and/or p24 genes. CCR5 expression was evaluated by RT-PCR and flow cytometry. The cells were infected with HIVBAL before or after transfection. Viral infection was assessed by flow cytometric analysis of intracellular p24 expression and p24 ELISA of culture supernatants. Viral RNA was detected.
Results: Application of p24 or CCR5 siRNA 2 days prior to infection reduced viral p24 expression and viral production 5-6 folds for up to 15 days after infection, a much longer time that reported in cell lines. This was accompanied by a comparable reduction in viral RNA levels. Combined targeting of both genes completely abrogated viral infection. As the interval between transfection and infection was raised from 2-15 days, the HIV suppressive effect of viral p24 siRNA progressively decreased and was eventually lost 10 days after infection. In contrast, viral inhibition by cellular CCR5 knockdown was uniformly sustained even when transfection preceded infection by 15 days. In agreement, Cy5-labeled p24 siRNA was only maintained up to 7 days in uninfected cells, while CCR5 ablation by the cognate siRNA was uniformly maintained for up to 20 days after transfection. However, when delivered into already infected macrophages, p24 siRNA was able to stably inhibit viral replication for up to 15 days.
Conclusions: These studies demonstrate that RNA interference has the potential to prevent and control HIV replication in non-dividing macrophages. The data also suggested that siRNAs might need target mRNAs for their intracellular sustenance.
