Targeted siRNA silencing of indoleamine 2, 3-dioxygenase in antigen-presenting cells using mannose-conjugated liposomes: a novel strategy for treatment of melanoma.

Indoleamine 2, 3-dioxygenase (IDO) expression in dendritic cells (DCs) leads to the inhibition of T-cell activation, induction of T-cell apoptosis, and promotion of T-cell differentiation into regulatory T cells. All of these could promote tumor escapement of the host's immune surveillance system. We hypothesized that DC-targeted gene silencing of IDO would enhance antitumor immunity and thus restrain tumor growth. Mannose receptors are highly expressed in antigen-presenting cells (APCs) including DCs. In this study, we developed a novel APC-targeted small interfering RNA delivery system using mannosed liposomes (Man-lipo) with encapsulated IDO small interfering RNA (Man-lipo-siIDO), which preferentially knocked down IDO expression in draining lymph node and spleen of melanoma-bearing mice. Mice treated with Man-lipo-siIDO displayed a delayed time of onset of implanted murine melanomas, increased survival time, reduced tumor size, and increased reactivity of T cells from spleen and lymph nodes against melanoma antigens. The enhanced antitumor immunity may be linked to inhibition of apoptosis in CD8 and CD4 T cells as well as Treg cells in spleen and lymph nodes. This study is the first to demonstrate that Man-lipo-siIDO can preferentially targets APCs and efficiently silence IDO expression in vitro and in vivo; events expected to enhance antitumor immune reactions against melanoma xenografts. This study supports the hypothesis that Man-lipo-siIDO may possess the potential for development as an immune-targeting therapeutic anticancer agent.

Prevention of hyperglycemia-induced myocardial apoptosis by gene silencing of Toll-like receptor-4.

BACKGROUND: Apoptosis is an early event involved in cardiomyopathy associated with diabetes mellitus. Toll-like receptor (TLR) signaling triggers cell apoptosis through multiple mechanisms. Up-regulation of TLR4 expression has been shown in diabetic mice. This study aimed to delineate the role of TLR4 in myocardial apoptosis, and to block this process through gene silencing of TLR4 in the myocardia of diabetic mice. METHODS: Diabetes was induced in C57/BL6 mice by the injection of streptozotocin. Diabetic mice were treated with 50 µg of TLR4 siRNA or scrambled siRNA as control. Myocardial apoptosis was determined by TUNEL assay. RESULTS: After 7 days of hyperglycemia, the level of TLR4 mRNA in myocardial tissue was significantly elevated. Treatment of TLR4 siRNA knocked down gene expression as well as diminished its elevation in diabetic mice. Apoptosis was evident in cardiac tissues of diabetic mice as detected by a TUNEL assay. In contrast, treatment with TLR4 siRNA minimized apoptosis in myocardial tissues. Mechanistically, caspase-3 activation was significantly inhibited in mice that were treated with TLR4 siRNA, but not in mice treated with control siRNA. Additionally, gene silencing of TLR4 resulted in suppression of apoptotic cascades, such as Fas and caspase-3 gene expression. TLR4 deficiency resulted in inhibition of reactive oxygen species (ROS) production and NADPH oxidase activity, suggesting suppression of hyperglycemia-induced apoptosis by TLR4 is associated with attenuation of oxidative stress to the cardiomyocytes. CONCLUSIONS: In summary, we present novel evidence that TLR4 plays a critical role in cardiac apoptosis. This is the first demonstration of the prevention of cardiac apoptosis in diabetic mice through silencing of the TLR4 gene.

Treatment of autoimmune arthritis using RNA interference-modulated dendritic cells.

Dendritic cells (DCs) have a dual ability to either stimulate or suppress immunity, which is primarily associated with the expression of costimulatory molecules. Ag-loaded DCs have shown encouraging clinical results for treating cancer and infectious diseases; however, the use of these cells as a means of suppressing immune responses is only recently being explored. Here, we describe the induction of RNA interference through administering short interfering RNA (siRNA) as a means of specifically generating tolerogenic DCs. Knockdown of CD40, CD80, and CD86, prior to loading DCs with the arthritogenic Ag collagen II, led to a population of cells that could effectively suppress onset of collagen-induced arthritis. Maximum benefits were observed when all three genes were concurrently silenced. Disease suppression was associated with inhibition of collagen II-specific Ab production and suppression of T cell recall responses. Downregulation of IL-2, IFN-gamma, TNF-alpha, and IL-17 and increased FoxP3(+) cells with regulatory activity were observed in collagen-induced arthritis mice treated with siRNA-transfected DCs. Collectively, these data support the use of ex vivo gene manipulation in DCs using siRNA to generate tailor-made tolerogenic vaccines for treating autoimmunity.

A novel allergen-specific therapy for allergy using CD40-silenced dendritic cells.

BACKGROUND: Induction of RNA interference with small interfering RNA (siRNA) has demonstrated therapeutic potential through the knockdown of target genes. We have previously reported that systemic administration of CD40 siRNA is capable of attenuating allergic symptoms but in an allergen-nonspecific fashion. However, siRNA-based allergen-specific therapy for allergy has not been developed. OBJECTIVE: We attempted to develop a new allergen-specific therapy for allergy using CD40-silenced and allergen-pulsed dendritic cells (DCs). METHODS: Bone marrow-derived DCs were silenced with CD40 siRNA and pulsed with ovalbumin (OVA). Mice had allergy after intraperitoneal sensitization with OVA and keyhole limpet hemocyanin, followed by intranasal challenge with the same allergens. The mice were treated with CD40-silenced and OVA-pulsed DCs (CD40-silenced OVA DCs) either before allergic sensitization or after establishing allergic rhinitis. RESULTS: Mice receiving CD40-silenced OVA DCs either before or after the establishment of allergic rhinitis showed remarkable reductions in allergic symptoms caused by OVA challenge, as well as anti-OVA IgE levels in sera. Additionally, CD40-silenced OVA DCs suppressed eosinophil infiltration at the nasal septum, OVA-specific T-cell responses, T-cell production of IL-4 and IL-5 after stimulation with OVA, and CD4(+)CD25(-) effector T-cell responses. Furthermore, CD40-silenced OVA DCs facilitated the generation of CD4(+)CD25(+) forkhead box protein 3-positive OVA-specific regulatory T cells, which inhibit allergic responses in vivo. However, CD40-silenced OVA DCs suppressed only OVA-specific allergy but did not inhibit keyhole limpet hemocyanin-induced allergy, suggesting that CD40-silenced OVA DCs induce allergen-specific tolerance. CONCLUSIONS: This study is the first to demonstrate a novel allergen-specific therapy for allergy through DC-mediated immune modulation after gene silencing of CD40.

RNAi-mediated CD40-CD154 interruption promotes tolerance in autoimmune arthritis.

INTRODUCTION: We have previously demonstrated that ex vivo inhibition of costimulatory molecules on antigen-pulsed dendritic cells (DCs) can be useful for induction of antigen-specific immune deviation and suppression of autoimmune arthritis in the collagen induced arthritis (CIA) model. The current study evaluated a practical method of immune modulation through temporary systemic inhibition of the costimulatory molecule CD40. METHODS: Mice with collagen II (CII)-induced arthritis (CIA) were administered siRNA targeting the CD40 molecule. Therapeutic effects were evaluated by clinical symptoms, histopathology, Ag-specific T cell and B cell immune responses. RESULTS: Systemic administration of CD40-targeting siRNA can inhibit antigen-specific T cell response to collagen II, as well as prevent pathogenesis of disease in both a pre- and post-immunization manner in the CIA model. Disease amelioration was associated with suppression of Th1 cytokines, attenuation of antibody production, and upregulation of T regulatory cells. CONCLUSIONS: These studies support the feasibility of transient gene silencing at a systemic level as a mechanism of resetting autoreactive immunity.