Less Is More: Novel Hepatocyte-Targeted siRNA Conjugates for Treatment of Liver-Related Disorders.

N-acetyl-galactosamine (GalNAc) conjugation enhances liver specificity for therapeutic oligonucleotides. Here we report on a novel design with improved activity and stability compared with a triantennary design. We applied a versatile monovalent serinol-GalNAc conjugation strategy. First, 1-4 serial serinol-linked GalNAc units were conjugated to terminal positions of small interfering RNA (siRNA) molecules. In primary hepatocytes, 5' antisense GalNAc conjugates were inactive, whereas 3' antisense and 3' or 5' sense conjugates displayed low activity for single GalNAc units, while 2-4 serial GalNAc conjugates were all equally potent. In mice, 5' sense conjugates with 2-4 serial GalNAc units were all as potent as a triantennary GalNAc control (1 mg/kg). Second, increased spacing between two serial 5' sense-conjugated GalNAc units did not affect in vitro activity. Finally, two single GalNAc units were positioned at opposite ends of the sense strand. A single dose (0.3 mg/kg) of this novel conjugate in mice showed a 3-fold reduction of serum target protein level at day 7 and 4-fold lower serum level at day 27, relative to an equimolar dose of a triantennary GalNAc conjugate of the same siRNA. Improved tritosome stability (by liquid chromatography-mass spectrometry [LC-MS] analysis) can at least partially explain the increased activity and duration of action for the novel GalNAc conjugate.

Reversible silencing of cytomegalovirus genomes by type I interferon governs virus latency.

Herpesviruses establish a lifelong latent infection posing the risk for virus reactivation and disease. In cytomegalovirus infection, expression of the major immediate early (IE) genes is a critical checkpoint, driving the lytic replication cycle upon primary infection or reactivation from latency. While it is known that type I interferon (IFN) limits lytic CMV replication, its role in latency and reactivation has not been explored. In the model of mouse CMV infection, we show here that IFNß blocks mouse CMV replication at the level of IE transcription in IFN-responding endothelial cells and fibroblasts. The IFN-mediated inhibition of IE genes was entirely reversible, arguing that the IFN-effect may be consistent with viral latency. Importantly, the response to IFNß is stochastic, and MCMV IE transcription and replication were repressed only in IFN-responsive cells, while the IFN-unresponsive cells remained permissive for lytic MCMV infection. IFN blocked the viral lytic replication cycle by upregulating the nuclear domain 10 (ND10) components, PML, Sp100 and Daxx, and their knockdown by shRNA rescued viral replication in the presence of IFNß. Finally, IFNß prevented MCMV reactivation from endothelial cells derived from latently infected mice, validating our results in a biologically relevant setting. Therefore, our data do not only define for the first time the molecular mechanism of IFN-mediated control of CMV infection, but also indicate that the reversible inhibition of the virus lytic cycle by IFNß is consistent with the establishment of CMV latency.

A new reporter mouse cytomegalovirus reveals maintained immediate-early gene expression but poor virus replication in cycling liver sinusoidal endothelial cells.

BACKGROUND: The MCMV major immediate early promoter/enhancer (MIEP) is a bidirectional promoter that drives the expression of the three immediate early viral genes, namely ie1, ie2 and ie3. The regulation of their expression is intensively studied, but still incompletely understood. METHODS: We constructed a reporter MCMV, (MCMV-MIEPr) expressing YFP and tdTomato under the control of the MIEP as proxies of ie1 and ie2, respectively. Moreover, we generated a liver sinusoidal endothelial cell line (LSEC-uniLT) where cycling is dependent on doxycycline. We used these novel tools to study the kinetics of MIEP-driven gene expression in the context of infection and at the single cell level by flow cytometry and by live imaging of proliferating and G0-arrested cells. RESULTS: MCMV replicated to higher titers in G0-arrested LSEC, and cycling cells showed less cytopathic effect or YFP and tdTomato expression at 5 days post infection. In the first 24 h post infection, however, there was no difference in MIEP activity in cycling or G0-arrested cells, although we could observe different profiles of MIEP gene expression in different cell types, like LSECs, fibroblasts or macrophages. We monitored infected LSEC-uniLT in G0 by time lapse microscopy over five days and noticed that most cells survived infection for at least 96 h, arguing that quick lysis of infected cells could not account for the spread of the virus. Interestingly, we noticed a strong correlation between the ratio of median YFP and tdTomato expression and length of survival of infected cells. CONCLUSION: By means of our newly developed genetic tools, we showed that the expression pattern of MCMV IE1 and IE2 genes differs between macrophages, endothelial cells and fibroblasts. Substantial and cell-cycle independent differences in the ie1 and ie2 transcription could also be observed within individual cells of the same population, and marked ie2 gene expression was associated with longer survival of the infected cells.

Leishmania promastigotes lack phosphatidylserine but bind annexin V upon permeabilization or miltefosine treatment.

The protozoan parasite Leishmania is an intracellular pathogen infecting and replicating inside vertebrate host macrophages. A recent model suggests that promastigote and amastigote forms of the parasite mimic mammalian apoptotic cells by exposing phosphatidylserine (PS) at the cell surface to trigger their phagocytic uptake into host macrophages. PS presentation at the cell surface is typically analyzed using fluorescence-labeled annexin V. Here we show that Leishmania promastigotes can be stained by fluorescence-labeled annexin V upon permeabilization or miltefosine treatment. However, combined lipid analysis by thin-layer chromatography, mass spectrometry and (31)P nuclear magnetic resonance (NMR) spectroscopy revealed that Leishmania promastigotes lack any detectable amount of PS. Instead, we identified several other phospholipid classes such phosphatidic acid, phosphatidylethanolamine; phosphatidylglycerol and phosphatidylinositol as candidate lipids enabling annexin V staining.

Ca2+-activated transbilayer movement of plasma membrane phospholipids in Leishmania donovani during ionomycin or thapsigargin stimulation.

The protozoan parasite Leishmania causes serious infections in humans all over the world. After being inoculated into the skin through the bite of an infected sandfly, Leishmania promastigotes must gain entry into macrophages to initiate a successful infection. Specific, surface exposed phospholipids have been implicated in Leishmania-macrophage interaction but the mechanisms controlling and regulating the plasma membrane lipid distribution remains to be elucidated. Here, we provide evidence for Ca(2+)-induced phospholipid scrambling in the plasma membrane of Leishmania donovani. Stimulation of parasites with ionomycin increases intracellular Ca(2+) levels and triggers exposure of phosphatidylethanolamine at the cell surface. We found that increasing intracellular Ca(2+) levels with ionomycin or thapsigargin induces rapid transbilayer movement of NBD-labelled phospholipids in the parasite plasma membrane that is bidirectional, independent of cellular ATP and not specific to the polar lipid head group. The findings suggest the presence of a Ca(2+)-dependent lipid scramblase activity in Leishmania parasites. Our studies further show that lipid scrambling is not activated by rapid exposure of promastigotes to higher physiological temperature that increases intracellular Ca(2+) levels.

Disruption of the lipid-transporting LdMT-LdRos3 complex in Leishmania donovani affects membrane lipid asymmetry but not host cell invasion.

Maintenance and regulation of the asymmetric lipid distribution across eukaryotic plasma membranes is governed by the concerted action of specific membrane proteins controlling lipid movement across the bilayer. Here, we show that the miltefosine transporter (LdMT), a member of the P4-ATPase subfamily in Leishmania donovani, and the Cdc50-like protein LdRos3 form a stable complex that plays an essential role in maintaining phospholipid asymmetry in the parasite plasma membrane. Loss of either LdMT or LdRos3 abolishes ATP-dependent transport of NBD-labelled phosphatidylethanolamine (PE) and phosphatidylcholine from the outer to the inner plasma membrane leaflet and results in an increased cell surface exposure of endogenous PE. We also find that promastigotes of L. donovani lack any detectable amount of phosphatidylserine (PS) but retain their infectivity in THP-1-derived macrophages. Likewise, infectivity was unchanged for parasites without LdMT-LdRos3 complexes. We conclude that exposure of PS and PE to the exoplasmic leaflet is not crucial for the infectivity of L. donovani promastigotes.