Christoph Coch

SiRNA delivery with exosome nanoparticles

A natural system for ferrying RNA between cells is used to transport siRNA to the mouse brain.

Exosomes as nucleic acid nanocarriers

Exosomes are nano-sized vesicles produced naturally by many cell types. They are specifically loaded with nucleic acid cargo, dependent on the exosome-producing cell and its homeostatic state. As natural intercellular shuttles of miRNA, exosomes influence an array of developmental, physiological and pathological processes in the recipient cell or tissue to which they can be selectively targeted by their tetraspanin surface-domains. By a review of current research, we illustrate here why exosomes are ideal nanocarriers for use in the targeted in vivo delivery of nucleic acids.

Sequence-specific activation of the DNA sensor cGAS by Y-form DNA structures as found in primary HIV-1 cDNA

Cytosolic DNA that emerges during infection with a retrovirus or DNA virus triggers antiviral type I interferon responses. So far, only double-stranded DNA (dsDNA) over 40 base pairs (bp) in length has been considered immunostimulatory. Here we found that unpaired DNA nucleotides flanking short base-paired DNA stretches, as in stem-loop structures of single-stranded DNA (ssDNA) derived from human immunodeficiency virus type 1 (HIV-1), activated the type I interferon–inducing DNA sensor cGAS in a sequence-dependent manner. DNA structures containing unpaired guanosines flanking short (12- to 20-bp) dsDNA (Y-form DNA) were highly stimulatory and specifically enhanced the enzymatic activity of cGAS. Furthermore, we found that primary HIV-1 reverse transcripts represented the predominant viral cytosolic DNA species during early infection of macrophages and that these ssDNAs were highly immunostimulatory. Collectively, our study identifies unpaired guanosines in Y-form DNA as a highly active, minimal cGAS recognition motif that enables detection of HIV-1 ssDNA.

Activation of Endothelial Toll-Like Receptor 3 Impairs Endothelial Function

Rationale: Endothelial dysfunction and atherosclerosis are chronic inflammatory diseases characterized by activation of the innate and acquired immune system. Specialized protein receptors of the innate immune system recognize products of microorganisms and endogenous ligands such as nucleic acids. Toll-like receptor 3 (TLR3), for example, detects long double-stranded RNA and is abundantly expressed in endothelial cells. Whether innate immunity contributes to atherogenic mechanisms in endothelial cells is poorly understood. Objective: We sought to determine the effects of TLR3 activation in endothelial cells. Methods and Results: We first investigated whether stimulation of TLR3 influences endothelial biology in mice. Intravenous injection of polyinosine polycytidylic acid, a synthetic double-stranded RNA analog and TLR3 ligand, impaired endothelium-dependent vasodilation, increased vascular production of reactive oxygen species, and reduced reendothelialization after carotid artery injury in wild-type mice compared with controls but had no effect in TLR3−/− animals. TLR3 stimulation not only induced endothelial dysfunction but also enhanced the formation of atherosclerotic plaques in apolipoprotein E–deficient mice. In vitro incubation of endothelial cells with polyinosine polycytidylic acid induced production of the proinflammatory cytokines interleukin-8 and interferon-&ggr;–induced protein 10, increased formation of reactive oxygen species, diminished proliferation, and increased apoptosis, which suggests that endothelial cells are able to directly detect and respond to TLR3 ligands. Neutralization of interleukin-8 and interferon-&ggr;–induced protein 10 antagonizes the observed negative effects of polyinosine polycytidylic acid. We found elevated levels of circulating endothelial progenitor cells in polyinosine polycytidylic acid–treated mice, although they displayed increased endothelial dysfunction. Stimulation of TLR3 in cultured endothelial progenitor cells, however, led to increased formation of reactive oxygen species, increased apoptosis, and reduced migration. Injection of endothelial progenitor cells that had been incubated with polyinosine polycytidylic acid ex vivo hindered reendothelialization after carotid artery injury. Therefore, endothelial progenitor cell function was affected by TLR3 stimulation. Finally, apolipoprotein E–deficient/TLR3-deficient mice exhibited improved endothelial function compared with apolipoprotein E–deficient/TLR3+/+ littermates. Conclusions: Immunorecognition of long double-stranded RNA by endothelial cells may be an important mechanism involved in endothelial cell activation and development of endothelial dysfunction.

Selective and direct activation of human neutrophils but not eosinophils by Toll-like receptor 8

BACKGROUND Granulocytes represent the largest fraction of immune cells in peripheral blood and are directly exposed to circulating Toll-like receptor (TLR) ligands. Although highly relevant for TLR-based therapies, because of the technical challenge, activation of the granulocyte subsets of neutrophils and eosinophils by TLR ligands is less well studied than activation of other immune cell subsets. OBJECTIVE The aim of this work was to study direct versus indirect neutrophil and eosinophil activation by TLR7 and TLR8 ligands. METHODS We used a new whole-blood assay, single cell-based cytokine detection, and highly purified primary human neutrophils and eosinophils to separate direct and indirect effects on these blood cell subsets. RESULTS We found indirect but not direct activation of neutrophils but not eosinophils in whole blood by using unmodified immunostimulatory RNA (isRNA; TLR7/8 ligand). In contrast, direct activation and stimulation of the respiratory burst and degranulation was seen with nuclease-stable isRNA and with the small-molecule TLR8 agonist 3M002 but not 3M001 (TLR7). Neutrophils expressed TLR8 but none of the other 2 RNA-detecting TLRs (TLR3 and TLR7). CONCLUSIONS Together, these results demonstrate that neutrophils are directly and fully activated through TLR8 but not TLR7. Furthermore, the results predict that the clinical utility of small-molecule TLR8 ligands or nuclease-stable RNA ligands for TLR8 might be limited because of neutrophil-mediated toxicity and that no such limitation applies for unmodified isRNA, which is known to induce desired T(H)1 activities in other immune cell subsets.

HIPEC ROC I: A phase i study of cisplatin administered as hyperthermic intraoperative intraperitoneal chemoperfusion followed by postoperative intravenous platinum‐based chemotherapy in patients with platinum‐sensitive recurrent epithelial ovarian cancer

This phase I study tested the safety, feasibility, pharmacokinetics and pharmacodynamics of cisplatin administered as hyperthermic intraoperative intraperitoneal chemoperfusion (HIPEC) in patients with platinum‐sensitive recurrent epithelial ovarian cancer (EOC) undergoing secondary cytoreductive surgery followed by postoperative platinum‐based intravenous chemotherapy. Twelve patients with operable, recurrent platinum‐sensitive EOC (recurrence ≥6 months after first‐line therapy) were included according to the classical 3+3 dose‐escalation design at three dose levels—60, 80 and 100 mg/m2. After surgical cytoreduction, a single dose of cisplatin was administered via HIPEC for 90 min at 41–43°C. Postoperatively, all patients were treated with standard intravenous platinum‐based combination chemotherapy. One of six patients experienced a dose‐limiting toxicity (grade 3 renal toxicity) at a dose of 100 mg/m2. The remaining five patients treated with 100 mg/m2 tolerated their treatment well. The recommended phase II dose was established at 100 mg/m2. The mean peritoneal‐to‐plasma AUC ratio was 19·5 at the highest dose level. Cisplatin‐induced DNA adducts were confirmed in tumor samples. Common postoperative grade 1–3 toxicities included fatigue, postoperative pain, nausea, and surgical site infection. The ability to administer standard intravenous platinum‐based chemotherapy after HIPEC was uncompromised. Cisplatin administered as HIPEC at a dose of 100 mg/m2 has an acceptable safety profile in selected patients undergoing secondary cytoreductive surgery for platinum‐sensitive recurrent EOC. Favorable pharmacokinetic and pharmacodynamic properties of HIPEC with cisplatin were confirmed at all dose levels, especially at 100 mg/m2. The results are encouraging to determine the efficacy of HIPEC as a complementary treatment in patients with EOC.

RIG-I detects triphosphorylated RNA of Listeria monocytogenes during infection in non-immune cells.

The innate immune system senses pathogens by pattern recognition receptors in different cell compartments. In the endosome, bacteria are generally recognized by TLRs; facultative intracellular bacteria such as Listeria, however, can escape the endosome. Once in the cytosol, they become accessible to cytosolic pattern recognition receptors, which recognize components of the bacterial cell wall, metabolites or bacterial nucleic acids and initiate an immune response in the host cell. Current knowledge has been focused on the type I IFN response to Listeria DNA or Listeria-derived second messenger c-di-AMP via the signaling adaptor STING. Our study focused on the recognition of Listeria RNA in the cytosol. With the aid of a novel labeling technique, we have been able to visualize immediate cytosolic delivery of Listeria RNA upon infection. Infection with Listeria as well as transfection of bacterial RNA induced a type-I-IFN response in human monocytes, epithelial cells or hepatocytes. However, in contrast to monocytes, the type-I-IFN response of epithelial cells and hepatocytes was not triggered by bacterial DNA, indicating a STING-independent Listeria recognition pathway. RIG-I and MAVS knock-down resulted in abolishment of the IFN response in epithelial cells, but the IFN response in monocytic cells remained unaffected. By contrast, knockdown of STING in monocytic cells reduced cytosolic Listeria-mediated type-I-IFN induction. Our results show that detection of Listeria RNA by RIG-I represents a non-redundant cytosolic immunorecognition pathway in non-immune cells lacking a functional STING dependent signaling pathway.

Immunogenic cell death of human ovarian cancer cells induced by cytosolic poly(I:C) leads to myeloid cell maturation and activates NK cells

Owing to high rates of tumor relapse, ovarian cancer remains a fatal disease for which new therapeutic approaches are urgently needed. Accumulating evidence indicates that immune stimulation may delay or even prevent disease recurrence in ovarian cancer. In order to elicit proinflammatory signals that induce or amplify antitumor immune reactivity, we mimicked viral infection in ascites‐derived ovarian cancer cells. By transfection or electroporation we targeted the synthetic double‐stranded RNA poly(I:C) intracellularly in order to activate melanoma differentiation‐associated gene‐5 (MDA‐5), a sensor of viral RNA in the cytosol of somatic cells. Cancer cells reacted with enhanced expression of HLA‐class I, release of CXCL10, IL‐6, and type I IFN as well as tumor cell apoptosis. Monocytes and monocyte‐derived DCs (MoDCs) engulfed MDA‐5‐activated cancer cells, and subsequently upregulated HLA‐class I/II and costimulatory molecules, and secreted CXCL10 and IFN‐α. Further, this proinflammatory milieu promoted cytolytic activity and IFN‐γ secretion of NK cells. Thus, our data suggest that the engagement of MDA‐5 in a whole tumor cell vaccine is a promising approach for the immunotherapy of ovarian cancer.

Higher activation of TLR9 in plasmacytoid dendritic cells by microbial DNA compared with self-DNA based on CpG-specific recognition of phosphodiester DNA

TLR9 detects DNA in endolysosomal compartments of human B cells and PDC. Recently, the concept of the CpG motif specificity of TLR9‐mediated detection, specifically of natural phosphodiester DNA, has been challenged. Unlike in human B cells, CpG specificity of natural phosphodiester DNA recognition in human PDC has not been analyzed in the literature. Here, we found that the induction of IFN‐α and TNF‐α in human PDC by phosphodiester ODNs containing one or two CG dinucleotides was reduced to a lower level when the CG dinucleotides were methylated and was abolished if the CGs were switched to GCs. Consistent with a high frequency of unmethylated CG dinucleotides, bacterial DNA induced high levels of IFN‐α in PDC; IFN‐α was reduced but not abolished upon methylation of bacterial DNA. Mammalian DNA containing low numbers of CG dinucleotides, which are frequently methylated, induced IFN‐α in PDC consistently but on a much lower level than bacterial DNA. For activation of PDC, phosphodiester ODNs and genomic DNA strictly required complexation with cationic molecules such as the keratinocyte‐derived antimicrobial peptide LL37 or a scrambled derivative. In conclusion, we demonstrate that self‐DNA complexed to cationic molecules activate PDC and thus, indeed, may function as DAMPs; nevertheless, the preference of PDC for CpG containing DNA provides the basis for the discrimination of microbial from self‐DNA even if DNA is presented in the condensed form of a complex.

Targeting the Cytosolic Innate Immune Receptors RIG‐I and MDA5 Effectively Counteracts Cancer Cell Heterogeneity in Glioblastoma

Cellular heterogeneity, for example, the intratumoral coexistence of cancer cells with and without stem cell characteristics, represents a potential root of therapeutic resistance and a significant challenge for modern drug development in glioblastoma (GBM). We propose here that activation of the innate immune system by stimulation of innate immune receptors involved in antiviral and antitumor responses can similarly target different malignant populations of glioma cells. We used short‐term expanded patient‐specific primary human GBM cells to study the stimulation of the cytosolic nucleic acid receptors melanoma differentiation‐associated gene 5 (MDA5) and retinoic acid‐inducible gene I (RIG‐I). Specifically, we analyzed cells from the tumor core versus “residual GBM cells” derived from the tumor resection margin as well as stem cell‐enriched primary cultures versus specimens without stem cell properties. A portfolio of human, nontumor neural cells was used as a control for these studies. The expression of RIG‐I and MDA5 could be induced in all of these cells. Receptor stimulation with their respective ligands, p(I:C) and 3pRNA, led to in vitro evidence for an effective activation of the innate immune system. Most intriguingly, all investigated cancer cell populations additionally responded with a pronounced induction of apoptotic signaling cascades revealing a second, direct mechanism of antitumor activity. By contrast, p(I:C) and 3pRNA induced only little toxicity in human nonmalignant neural cells. Granted that the challenge of effective central nervous system (CNS) delivery can be overcome, targeting of RIG‐I and MDA5 could thus become a quintessential strategy to encounter heterogeneous cancers in the sophisticated environments of the brain. STEM Cells 2013;31:1064–1074