Intranasal Delivery of Immunotherapeutic Nanoformulations for Treatment of Glioma Through in situ Activation of Immune Response.

PURPOSE: Some chemotherapeutics have been shown to induce both the release of damage-associated molecular patterns (DAMPs) and the production of type I interferon (IFN-I), leading to immunogenic cell death (ICD). However, the standard chemotherapy drug for glioma, temozolomide (TMZ), cannot induce ICD as it cannot activate IFN-I signaling. Moreover, inefficient delivery of immunostimulants across the blood-brain barrier (BBB) is the main obstacle to overcome in order to induce local immune responses in the brain. METHODS: A new oligonucleotide nanoformulation (Au@PP)/poly(I:C)) was constructed by coating gold nanoparticles (AuNPs) with methoxypolyethylene glycol (mPEG)-detachable (d)-polyethyleneimine (PEI) (Au@PP) followed by inducing the formation of electrostatic interactions with polyinosinic-polycytidylic acid (poly(I:C)). Intracranial GL261 tumor-bearing C57BL/6 mice were used to explore the therapeutic outcomes of Au@PP/poly(I:C) plus TMZ in vivo. The anti-tumor immune response in the brain induced by this treatment was analyzed by RNA sequencing and immunohistochemical analyses. RESULTS: Au@PP/poly(I:C) induced IFN-I production after endocytosis into glioma cells in vitro. Additionally, Au@PP/poly(I:C) was efficiently accumulated in the glioma tissue after intranasal administration, which allowed the nanoformulation to enter the brain while bypassing the BBB. Furthermore, Au@PP/poly(I:C) plus TMZ significantly improved the overall survival of the tumor-bearing mice compared with group TMZ only. RNA sequencing and immunohistochemical analyses revealed efficient immune response activation and T lymphocyte infiltration in the Au@PP/poly(I:C) plus TMZ group. CONCLUSION: This study demonstrates that intranasal administration of Au@PP/poly(I:C) combined with TMZ induces ICD, thereby stimulating an in situ immune response to inhibit glioma growth.

Novel methods for nucleotide length control in double-stranded polyinosinic-polycytidylic acid production using uneven length components.

Polyinosinic-polycytidylic acid (PIC), a double-stranded RNA that induces innate immunity in mammals, is a candidate immunopotentiator for pharmaceuticals. The potency and adverse effects of PIC are strongly correlated with the nucleotide length, and the inability to precisely control the length in PIC production limits its practical use. Length extension during the annealing process is the major factor underlying the lack of control, but tuning the annealing conditions is insufficient to resolve this issue. In this study, we developed a novel method to produce accurate nucleotide length PIC at an industrial scale. The length extension was significantly suppressed by the assembly of multiple short polyinosinic acid molecules with one long polycytidylic acid molecule. A newly developed PIC, uPIC100-400, demonstrated a reproducible length and better storage stability than that of corresponding evenly structured PIC. Human dsRNA receptors exhibited equivalent responsiveness to uPIC100-400 and the evenly structured PIC with the same length.

Intron-containing type I and type III IFN coexist in amphibians: refuting the concept that a retroposition event gave rise to type I IFNs.

Type I and III IFNs are structurally related cytokines with similar antiviral functions. They have different genomic organizations and bind to distinct receptor complexes. It has been vigorously debated whether the recently identified intron containing IFN genes in fish and amphibians belong to the type I or III IFN family or diverged from a common ancestral gene, that subsequently gave rise to both types. In this report, we have identified intron containing type III IFN genes that are tandemly linked in the Xenopus tropicalis genome and hence demonstrate for the first time that intron containing type I and III genes diverged relatively early in vertebrate evolution, and at least by the appearance of early tetrapods, a transition period when vertebrates migrated from an aquatic environment to land. Our data also suggest that the intronless type I IFN genes seen in reptiles, birds, and mammals have originated from a type I IFN transcript via a retroposition event that led to the disappearance of intron-containing type I IFN genes in modern vertebrates. In vivo and in vitro studies in this paper show that the Xenopus type III IFNs and their cognate receptor are ubiquitously expressed in tissues and primary splenocytes and can be upregulated by stimulation with synthetic double-stranded RNA, suggesting they are involved in antiviral defense in amphibians.

Protein kinase R mediates intestinal epithelial gene remodeling in response to double-stranded RNA and live rotavirus.

As sentinels of host defense, intestinal epithelial cells respond to the viral pathogen rotavirus by activating a gene expression that promotes immune cell recruitment and activation. We hypothesized that epithelial sensing of rotavirus might target dsRNA, which can be detected by TLR3 or protein kinase R (PKR). Accordingly, we observed that synthetic dsRNA, polyinosinic acid:cytidylic acid (poly(I:C)), potently induced gene remodeling in model intestinal epithelia with the specific pattern of expressed genes, including both classic proinflammatory genes (e.g., IL-8), as well as genes that are classically activated in virus-infected cells (e.g., IFN-responsive genes). Poly(I:C)-induced IL-8 was concentration dependent (2-100 mug/ml) and displayed slower kinetics compared with IL-8 induced by bacterial flagellin (ET(50) approximately 24 vs 8 h poly(I:C) vs flagellin, respectively). Although model epithelia expressed detectable TLR3 mRNA, neither TLR3-neutralizing Abs nor chloroquine, which blocks activation of intracellular TLR3, attenuated epithelial responses to poly(I:C). Conversely, poly(I:C)-induced phosphorylation of PKR and inhibitors of PKR, 2-aminopurine and adenine, ablated poly(I:C)-induced gene expression but had no effect on gene expression induced by flagellin, thus suggesting that intestinal epithelial cell detection of dsRNA relies on PKR. Consistent with poly(I:C) detection by an intracellular molecule such as PKR, we observed that both uptake of and responses to poly(I:C) were polarized to the basolateral side. Lastly, we observed that the pattern of pharmacologic inhibition of responses to poly(I:C) was identical to that seen in response to infection by live rotavirus, indicating a potentially important role for PKR in activating intestinal epithelial gene expression in rotavirus infection.

8-Azido double-stranded RNA photoaffinity probes. Enzymatic synthesis, characterization, and biological properties of poly(I,8-azidoI).poly(C) and poly(I,8-azidoI).poly(C12U) with 2',5'-oligoadenylate synthetase and protein kinase.

The technique of photoaffinity labeling has been applied to the double-stranded RNA (dsRNA)-dependent enzyme 2',5'-oligoadenylate (2-5A) synthetase to provide a means for the examination of RNA-protein interaction(s) in the dsRNA allosteric binding domain of this enzyme. The synthesis, characterization, and biological properties of the photoaffinity probe poly[( 32P]I,8-azidoI).poly(C) and its mismatched analog poly[( 32P]I,8-azidoI).poly(C12U), which mimic the parent molecules poly(I).poly(C) and poly(I).poly(C12U), are described. The efficacy of poly[( 32P]I,8-azidoI).poly(C) and poly[( 32P]I,8-azidoI).poly(C12U) as allosteric site-directed activators is demonstrated using highly purified 2-5A synthetase from rabbit reticulocyte lysates and from extracts of interferon-treated HeLa cells. The dsRNA photoprobes activate these two 2-5A synthetases. Saturation of 2-5A synthetase is observed at 6 x 10(-4) g/ml poly[( 32P]I,8-azidoI).poly(C) following photolysis for 20 s at 0 degrees C. The photoincorporation of poly[( 32P]I,8-azidoI).poly(C) is specific, as demonstrated by the prevention of photoincorporation by native poly(I).poly(C). DNA, poly(I), and poly(C) are not competitors of poly[( 32P]I,8-azidoI).poly(C). Following UV irradiation of 2-5A synthetase with poly[( 32P]I,8-azidoI).poly(C), the reaction mixture is treated with micrococcal nuclease to hydrolyze azido dsRNA that is not cross-linked to the enzyme. A radioactive band of 110 kDa (the same as that reported for native rabbit reticulocyte lysate 2-5A synthetase) is observed following sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. The specific photolabeling of the 2-5A synthetase suggests that the azido dsRNA is intrinsic to the allosteric binding domain. The utility of poly[( 32P]I,8-azidoI).poly(C) for the detection of dsRNA-dependent binding proteins and the isolation of peptides at or near the allosteric binding site is discussed.

Purification of carboxymethylcellulose decreases toxicity of poly ICLC in mice.

The interferon inducer poly ICLC has toxic side effects; the carboxymethylcellulose (CMC) component is a possible source of this toxicity. We have purified CMC by ethanol extraction, and found that poly ICLC made with purified CMC is not significantly different in its effectiveness as an interferon inducer in mice than poly ICLC made with unpurified CMC, but is less toxic.