Background:  Phosphorodiamidate morpholino oligomers (PMO) are synthetic antisense molecules that interfere with translation, pre-mRNA splicing, and RNA synthesis by sterically blocking complementary RNA sequences. PMO conjugated to arginine-rich cell-penetrating peptides (PPMO) are efficiently delivered to leukocytes including T cells and dendritic cells. PPMO targeting the human interleukin-10 (IL-10) gene were tested for the ability to modulate IL-10 expression and alter the immunosuppressive activity of this cytokine.

Methods:  A series of PPMO that target the human IL-10 start codon and pre-mRNA splice acceptor sites were evaluated by treating peripheral blood mononuclear cell (PBMC) -derived human dendritic cells in culture. Altered splicing of the IL-10 pre-mRNA was evaluated by polymerase chain reaction (PCR). PPMO-treated (5 µM) dendritic cells (DC) stimulated with lipopolysaccharide (LPS) (10 and 100 ng/mL), bacterial cell wall extract (30 µg/mL), and Zymosan (30 µg/mL) were evaluated for IL-10 secretion using an IL-10 enzyme-linked immunosorbent spot (ELISpot) assay. Mice (BALB/c and C57BL/6) were treated with 50 µg PPMO targeting a murine IL-10 splice acceptor site and subsequently challenged with a lethal inoculum (1000 pfu) of Ebola or Marburg virus, respectively.

Results:  PPMO that effectively induced altered splicing of IL-10 pre-mRNA were selected by their ability to induce alternatively spliced mRNA in vitro. Compared to control PPMO, a specific PPMO inhibited IL-10 secretion by 80 to 90% from human DC stimulated with LPS, bacterial cell wall extract or Zymosan. Lethal murine filovirus infection models were used to determine the antiviral efficacy of anti-IL-10 PPMO. Anti-IL-10 PPMO protected 90% and 80% of mice (n = 10) from lethal doses of Marburg virus and Ebola virus, respectively.

Conclusions:  PPMO that effectively knockdown IL-10 gene expression in dendritic cells in culture were also shown to be an effective prophylactic in lethal in vivo filovirus challenge models. One explanation for this result is that knockdown of IL-10 provides a more robust adaptive Th1 response. IL-10 has been implicated as a contributing factor in the immunosuppression observed in patients chronically infected with a number of pathogens including hepatitis C virus (HCV), Mycobacterium tuberculosis, and HIV. Furthermore, IL-10-mediated suppression of CD4⁺ T cell activation may be a limiting factor in establishing protective immunity by HIV-1 vaccines. Anti-IL-10 PPMO may therefore represent useful molecular adjuvants for either DC-based immunotherapy or protective vaccine candidates.