In vitro: The jetOPTIMUS transfection reagent is specifically designed for in vitro transfection of hard-to-transfect cells, including primary cells/blood cells. Whole blood from animals M8001 and F8003 was drawn into hirudin-coated blood collection tubes. A total of 185 µL of blood was then transferred into the wells of a 96-well round bottom plate and incubated for 6 hours with 15 µL of jetOPTIMUS transfection reagent containing poly(dA:dT). As an untreated control, blood samples were incubated with transfection reagent only. Thereafter, blood was collected from 5 wells of treated or untreated control samples and combined, cells were lysed with RLT buffer containing 1% β-mercaptoethanol, and total RNA was isolated using the QIAamp RNA Blood Mini kit. The mRNA was subsequently converted to complementary (c) DNA and the expression of receptors, adaptor molecules, and effector cytokines was determined by real time PCR assay, as described recently (38, 40). A fold-change of ≥2.1 from the untreated control was considered a positive result for increased gene expression after in vitro treatment with poly(dA:dT). Compared to the transfection control, blood cells of both woodchucks treated with poly(dA:dT) showed increased expression of receptors (fold-change: ZBP1/DAI, M8001, 21.3, F8003, 19.8; AIM2, M8001, 4.4, F8003, 2.7) and effector cytokines, except for IL-18 in M8001 (fold-change: IFN-β, M8001, 56.9, F8003, 44.0; IL-1β, M8001, 2.1, F8003, 2.6; IL-18, M8001, 1.2, F8003, 2.7). The expression of adaptor molecules stayed close to the transfection control baseline, expect for ASC in blood cells of M8001 (fold-change: 14.9). In vivo: The two WHV-uninfected adult woodchucks, M8001 and F8003, were confirmed negative for serum WHV DNA, WHV surface antigen (WHsAg), and antibodies to WHsAg (anti-WHs antibodies) using assays described previously. Both animals were intravenously injected with a single dose of poly(dA:dT) mixed in in vivo-jetPEI-Gal transfection reagent (PolyPlus Transfection). Woodchuck M8001 received a low dose (125 µg/kg), while animal F8003 received a high dose (375 µg/kg) of poly(dA:dT). Blood samples were collected into PAXGene tubes at pre-treatment, and then at 24-hours post-treatment for analyzing gene expression. Ultrasound-guided, percutaneous liver biopsies were also obtained at pre-treatment and at 24-hours post-treatment, placed immediately in liquid nitrogen, and stored at 80°C for subsequent gene expression analysis. Body weights, body temperatures, hematology, clinical chemistry, and liver histology of both woodchucks were frequently determined. Single dose administration of poly(dA:dT) to woodchucks results in agonistic activation of ZBP1/DAI and AIM2 receptor pathways in the liver and periphery. Liver biopsies and blood from animals M8001 and F8003 were collected 24 hours after intravenous injection of a single low (M8001) or high (F8003) dose of poly(dA:dT) in in vivo-jetPEI-Gal transfection reagent. The fold-changes in transcript level of receptors (ZBP1/DAI and AIM2) and effector cytokines (IFN-β, IL-18, and IL-1β) in (A) liver and (B) blood are shown, when compared to their transcript level at pre-treatment in liver biopsies and blood from the same animals, which was set at 1.0. FC, fold-change. Single dose administration of poly(dA:dT) to woodchucks results in an increased number of IFN-γ secreting blood cells and nearly unchanged liver enzymes. (A) PBMCs from animals M8001 and F8003 were isolated 24 hours after intravenous injection of a single low (M8001) or high (F8003) dose of poly(dA:dT) in in vivo-jetPEI-Gal transfection reagent and subjected to an ELISpot assay. The fold-changes in spot-forming units (SFU) are shown, when compared to the spot number at pre-treatment in PBMCs from both animals, which was set at 1.0. (B) The levels of ALT, AST, SDH, and GGT in serum of both animals measured prior to treatment and at 24 hours post-treatment are shown. The normal range of liver enzyme levels in WHV-uninfected woodchucks is 1-6 IU/mL for ALT, 9-55 IU/mL for AST, and 0.8-2.6 IU/mL for GGT (46). For SDH, the normal range in WHV-uninfected woodchucks is 7-31 IU/mL (47). FC, fold-change; IU, international units As a first step in this direction, administration of a single dose of poly(dA:dT) to WHV-uninfected woodchucks was safe, based on unchanged body weight, body temperature, hematology, and clinical chemistry 24 hours later. Furthermore, changes in liver histology were absent and no marked elevation in liver enzymes was observed, except for some minor increases in GGT and SDH in one or both animals, respectively. Following treatment, the intrahepatic expression of ZBP1/DAI and AIM2 receptors and their effector cytokines increased in the liver and periphery of both woodchucks, but the changes did not appear dose-dependent. Consistent with the role of IL-18 as a co-activator of IFN-γ production (44), a dose-dependent increase in T-cells secreting IFN-γ was observed. Overall, the present study demonstrated an antiviral benefit that is associated with the agonistic activation of more than one PRR pathway. More specifically, the activation of one cytosolic DNA (i.e., ZBP1/DAI) and one inflammasome (i.e., AIM2) receptor pathway has the advantage of inducing effector cytokines with rather distinct functions that could promote an overall more suitable immune response against chronic HBV infection. The safe in vivo administration of poly(dA:dT) that resulted in the intrahepatic upregulation of both receptors further encourages evaluating this agonist for therapeutic efficacy against CHB.