Retinoic acid-inducible gene-I (RIG-I) is a cytoplasmic immune receptor sensing viral RNA. It triggers the release of type I interferons (IFN) and proinflammatory cytokines inducing an adaptive ...cellular immune response. We investigated the therapeutic potential of systemic RIG-I activation by short 5'-triphosphate-modified RNA (ppp-RNA) for the treatment of acute myeloid leukemia (AML) in the syngeneic murine C1498 AML tumor model. ppp-RNA treatment significantly reduced tumor burden, delayed disease onset and led to complete remission including immunological memory formation in a substantial proportion of animals. Therapy-induced tumor rejection was dependent on CD4
and CD8
T cells, but not on NK or B cells, and relied on intact IFN and mitochondrial antiviral signaling protein (MAVS) signaling in the host. Interestingly, ppp-RNA treatment induced programmed death ligand 1 (PD-L1) expression on AML cells and established therapeutic sensitivity to anti-PD-1 checkpoint blockade in vivo. In immune-reconstituted humanized mice, ppp-RNA treatment reduced the number of patient-derived xenografted (PDX) AML cells in blood and bone marrow while concomitantly enhancing CD3
T cell counts in the respective tissues. Due to its ability to establish a state of full remission and immunological memory, our findings show that ppp-RNA treatment is a promising strategy for the immunotherapy of AML.
DNA-based nanostructures have received great attention as molecular vehicles for cellular delivery of biomolecules and cancer drugs. Here, we report on the cellular uptake of tubule-like DNA ...tile-assembled nanostructures 27 nm in length and 8 nm in diameter that carry siRNA molecules, folic acid and fluorescent dyes. In our observations, the DNA structures are delivered to the endosome and do not reach the cytosol of the
-expressing HeLa cells that were used in the experiments. Consistent with this observation, no elevated silencing of the
gene could be detected. Furthermore, the presence of up to six molecules of folic acid on the carrier surface did not alter the uptake behavior and gene silencing. We further observed several challenges that have to be considered when performing
and
experiments with DNA structures: (i) DNA tile tubes consisting of 42 nt-long oligonucleotides and carrying single- or double-stranded extensions degrade within one hour in cell medium at 37 °C, while the same tubes without extensions are stable for up to eight hours. The degradation is caused mainly by the low concentration of divalent ions in the media. The lifetime in cell medium can be increased drastically by employing DNA tiles that are 84 nt long. (ii) Dyes may get cleaved from the oligonucleotides and then accumulate inside the cell close to the mitochondria, which can lead to misinterpretation of data generated by flow cytometry and fluorescence microscopy. (iii) Single-stranded DNA carrying fluorescent dyes are internalized at similar levels as the DNA tile-assembled tubes used here.
The incidence of allergic diseases has increased over the past 50 years, likely due to environmental factors. However, the nature of these factors and the mode of action by which they induce the type ...2 immune deviation characteristic of atopic diseases remain unclear. It has previously been reported that dietary sodium chloride promotes the polarization of T helper 17 (T
17) cells with implications for autoimmune diseases such as multiple sclerosis. Here, we demonstrate that sodium chloride also potently promotes T
2 cell responses on multiple regulatory levels. Sodium chloride enhanced interleukin-4 (IL-4) and IL-13 production while suppressing interferon-γ (IFN-γ) production in memory T cells. It diverted alternative T cell fates into the T
2 cell phenotype and also induced de novo T
2 cell polarization from naïve T cell precursors. Mechanistically, sodium chloride exerted its effects via the osmosensitive transcription factor NFAT5 and the kinase SGK-1, which regulated T
2 signature cytokines and master transcription factors in hyperosmolar salt conditions. The skin of patients suffering from atopic dermatitis contained elevated sodium compared to nonlesional atopic and healthy skin. These results suggest that sodium chloride represents a so far overlooked cutaneous microenvironmental checkpoint in atopic dermatitis that can induce T
2 cell responses, the orchestrators of atopic diseases.
The prognosis of acute myeloid leukemia (AML) is poor due to frequent relapse after initial remission. The development of new approaches to postremission therapy for elimination of minimal residual ...disease remains a major scientific and clinical challenge. We strive to combine two different innovative therapeutic concepts to develop a new specific and personalized treatment for AML. siRNAs are used to knock down either a gene that drives leukemogenesis due to genetic alterations in specific cases of AML (e.g., FLT3, NPM1) or a gene that is essential for the survival of the leukemic cells (e.g., BRD4, MCL1, PLK1). By adding a triphosphate modification to the 5' end, the siRNA molecules additionally become ligands for the cytosolic pattern recognition receptor RIG-I (retinoic acid inducible gene I). Its activation mimics viral infection and leads to the production of inflammatory cytokines and induction of apoptosis in the target cell. We expect these bifunctional molecules to result in a decrease of viable AML cells and in the induction of an immune response similar to an active immunization.
This concept was successfully tested in vitro for several target genes in AML cell lines. We could demonstrate that the specific gene knockdown leads to inhibited proliferation, increased apoptosis and higher sensitivity to chemotherapeutic agents. Activation of RIG-I by triphosphate-modified RNA additionally stimulated an inflammatory response by the leukemic cells and increased the apoptosis rate.
A major hurdle for all siRNA-based anti-cancer strategies is the specific delivery of the RNA into tumor cells. In vivo liposomal transfection of siRNA molecules has been used in various tumor models, but generally results in ineffective and unspecific delivery. We are testing DNA-based nanoparticles coupled with molecules that target receptors specific for or overexpressed on AML cells. By coupling bifunctional siRNA molecules to these nanoparticles, they should be efficiently and selectively transported into the cytosol of AML cells.
Proof-of-concept in vivo studies in AML mouse models are in preparation. The long-term goal of this project is the development of a set of bifunctional siRNA molecules for the individualized treatment of AML.
No relevant conflicts of interest to declare.
DNA-based nanostructures have received great attention as molecular vehicles for cellular delivery of biomolecules and cancer drugs. Here, we report on the cellular uptake of tubule-like DNA ...tile-assembled nanostructures 27 nm in length and 8 nm in diameter that carry siRNA molecules, folic acid and fluorescent dyes. In our observations, the DNA structures are delivered to the endosome and do not reach the cytosol of the GFP-expressing HeLa cells that were used in the experiments. Consistent with this observation, no elevated silencing of the GFP gene could be detected. Furthermore, the presence of up to six molecules of folic acid on the carrier surface did not alter the uptake behavior and gene silencing. We further observed several challenges that have to be considered when performing in vitro and in vivo experiments with DNA structures: (i) DNA tile tubes consisting of 42 nt-long oligonucleotides and carrying single- or double-stranded extensions degrade within one hour in cell medium at 37 °C, while the same tubes without extensions are stable for up to eight hours. The degradation is caused mainly by the low concentration of divalent ions in the media. The lifetime in cell medium can be increased drastically by employing DNA tiles that are 84 nt long. (ii) Dyes may get cleaved from the oligonucleotides and then accumulate inside the cell close to the mitochondria, which can lead to misinterpretation of data generated by flow cytometry and fluorescence microscopy. (iii) Single-stranded DNA carrying fluorescent dyes are internalized at similar levels as the DNA tile-assembled tubes used here.
Serum antibodies against myelin-oligodendrocyte-glycoprotein (MOG-IgG) are detectable in a proportion of patients with acute or relapsing neuroinflammation. It is unclear, if neuro-axonal damage ...occurs only in an attack-dependent manner or also progressively. Therefore, this study aimed to investigate longitudinally intra-retinal layer changes in eyes without new optic neuritis (ON) in MOG-IgG-seropositive patients.
We included 38 eyes of 24 patients without ON during follow-up (F/U) median years (IQR) 1.9 (1.0-2.2) and 56 eyes of 28 age- and sex-matched healthy controls (HC). The patient group's eyes included 18 eyes without (Eye
) and 20 eyes with history of ON (Eye
). Using spectral domain optical coherence tomography (OCT), we acquired peripapillary retinal nerve fiber layer thickness (pRNFL) and volumes of combined ganglion cell and inner plexiform layer (GCIP), inner nuclear layer (INL), and macular volume (MV). High-contrast visual acuity (VA) was assessed at baseline.
At baseline in Eye
, pRNFL (94.3 ± 15.9 μm, p = 0.36), INL (0.26 ± 0.03 mm
, p = 0.11), and MV (2.34 ± 0.11 mm
, p = 0.29) were not reduced compared to HC; GCIP showed thinning (0.57 ± 0.07 mm
; p = 0.008), and VA was reduced (logMAR 0.05 ± 0.15 vs. - 0.09 ± 0.14, p = 0.008) in comparison to HC. Longitudinally, we observed pRNFL thinning in models including all patient eyes (annual reduction - 2.20 ± 4.29 μm vs. - 0.35 ± 1.17 μm, p = 0.009) in comparison to HC. Twelve Eye
with other than ipsilateral ON attacks ≤ 6 months before baseline showed thicker pRNFL at baseline and more severe pRNFL thinning in comparison to 6 Eye
without other clinical relapses.
We observed pRNFL thinning in patients with MOG-IgG during F/U, which was not accompanied by progressive GCIP reduction. This effect could be caused by a small number of Eye
with other than ipsilateral ON attacks within 6 months before baseline. One possible interpretation could be a reduction of the swelling, which could mean that MOG-IgG patients show immune-related swelling in the CNS also outside of an attack's target area.
BackgroundSerum antibodies against myelin-oligodendrocyte-glycoprotein (MOG-IgG) are detectable in a proportion of patients with acute or relapsing neuroinflammation. It is unclear, if neuro-axonal ...damage occurs only in an attack-dependent manner or also progressively. Therefore, this study aimed to investigate longitudinally intra-retinal layer changes in eyes without new optic neuritis (ON) in MOG-IgG-seropositive patients.MethodsWe included 38 eyes of 24 patients without ON during follow-up (F/U) median years (IQR) 1.9 (1.0-2.2) and 56 eyes of 28 age- and sex-matched healthy controls (HC). The patient group's eyes included 18 eyes without (Eye(ON-)) and 20 eyes with history of ON (Eye(ON+)). Using spectral domain optical coherence tomography (OCT), we acquired peripapillary retinal nerve fiber layer thickness (pRNFL) and volumes of combined ganglion cell and inner plexiform layer (GCIP), inner nuclear layer (INL), and macular volume (MV). High-contrast visual acuity (VA) was assessed at baseline.ResultsAt baseline in Eye(ON-), pRNFL (94.315.9 mu m, p=0.36), INL (0.26 +/- 0.03mm(3), p=0.11), and MV (2.34 +/- 0.11mm(3), p=0.29) were not reduced compared to HC;GCIP showed thinning (0.57 +/- 0.07mm(3);p=0.008), and VA was reduced (logMAR 0.05 +/- 0.15 vs. -0.09 +/- 0.14, p=0.008) in comparison to HC. Longitudinally, we observed pRNFL thinning in models including all patient eyes (annual reduction -2.20 +/- 4.29 mu m vs. -0.35 +/- 1.17 mu m, p=0.009) in comparison to HC. Twelve Eye(ON-) with other than ipsilateral ON attacks <= 6months before baseline showed thicker pRNFL at baseline and more severe pRNFL thinning in comparison to 6 Eye(ON-) without other clinical relapses.Conclusion We observed pRNFL thinning in patients with MOG-IgG during F/U, which was not accompanied by progressive GCIP reduction. This effect could be caused by a small number of Eye(ON-) with other than ipsilateral ON attacks within 6months before baseline. One possible interpretation could be a reduction of the swelling, which could mean that MOG-IgG patients show immune-related swelling in the CNS also outside of an attack's target area.
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