Arnold Grünweller

MicroRNA Replacement Therapy for miR-145 and miR-33a Is Efficacious in a Model of Colon Carcinoma

MicroRNAs (miRNA) aberrantly expressed in tumors may offer novel therapeutic approaches to treatment. miR-145 is downregulated in various cancers including colon carcinoma in which in vitro studies have established proapoptotic and antiproliferative roles. miR-33a was connected recently to cancer through its capacity to downregulate the oncogenic kinase Pim-1. To date, miRNA replacement therapy has been hampered by the lack of robust nonviral delivery methods for in vivo administration. Here we report a method of miRNA delivery by using polyethylenimine (PEI)-mediated delivery of unmodified miRNAs, using miR-145 and miR-33a to preclinically validate the method in a mouse model of colon carcinoma. After systemic or local application of low molecular weight PEI/miRNA complexes, intact miRNA molecules were delivered into mouse xenograft tumors, where they caused profound antitumor effects. miR-145 delivery reduced tumor proliferation and increased apoptosis, with concomitant repression of c-Myc and ERK5 as novel regulatory target of miR-145. Similarly, systemic injection of PEI-complexed miR-33a was validated as a novel therapeutic targeting method for Pim-1, with antitumor effects comparable with PEI/siRNA-mediated direct in vivo knockdown of Pim-1 in the model. Our findings show that chemically unmodified miRNAs complexed with PEI can be used in an efficient and biocompatible strategy of miRNA replacement therapy, as illustrated by efficacious delivery of PEI/miR-145 and PEI/miR-33a complexes in colon carcinoma.

The proto-oncogene Pim-1 is a target of miR-33a

The constitutively active serine/threonine kinase Pim-1 is upregulated in different cancer types, mainly based on the action of several interleukines and growth factors at the transcriptional level. So far, a regulation of oncogenic Pim-1 by microRNAs (miRNAs) has not been reported. Here, we newly establish miR-33a as a miRNA with potential tumor suppressor activity, acting through inhibition of Pim-1. A screen for miRNA expression in K562 lymphoma, LS174T colon carcinoma and several other cell lines revealed generally low endogenous miR-33a levels relative to other miRNAs. Transfection of K562 and LS174T cells with a miR-33a mimic reduced Pim-1 levels substantially. In contrast, the cell-cycle regulator cyclin-dependent kinase 6 predicted to be a conserved miR-33a target, was not downregulated by the miR-33a mimic. Seed mutagenesis of the Pim-1 3′-untranslated region in a luciferase reporter construct and in a Pim-1 cDNA expressed in Pim-1-deficient Skov-3 cells demonstrated specific and direct downregulation of Pim-1 by the miR-33a mimic. The persistence of this effect was comparable to that of a small interfering RNA-mediated knockdown of Pim-1, resulting in decelerated cell proliferation. In conclusion, we demonstrate the potential of miR-33a to act as a tumor suppressor miRNA, which suggests miR-33a replacement therapy through delivery of miR mimics as a novel therapeutic strategy.

RNA interference as a gene-specific approach for molecular medicine.

The discovery of RNA interference (RNAi) in eukaryotic cells has been the major recent breakthrough in molecular and cell biology. RNAi machineries exert biological functions in gene regulation, genome defense and chromatin architecture and dynamics. The potential of RNAi to silence any gene of interest in a highly specific and efficient manner via double-stranded RNA (dsRNA) has literally revolutionized modern genetics. RNAi-based functional genomics now permits, for the first time, to evaluate the cellular role of individual gene products on a genome-wide scale in higher organisms like mammals, presenting an alternative to the generation of animal knockouts often doomed to failure because of a lethal phenotype. RNAi has had an enormous impact on the development of novel disease models in animals, and it is likely that small interfering RNAs (siRNAs), which are the trigger molecules for RNA silencing, will become an invaluable tool for the treatment of genetic diseases. First clinical trials, using siRNAs directed against the vascular endothelial growth factor (VEGF) or one of its receptors, have been initiated recently for the treatment of age-related macular degeneration. Improving guidelines for the rational design of siRNAs, based on recent progress in understanding the mechanisms underlying RNAi, as well as the introduction of chemical modifications into siRNAs are expected to improve their pharmacokinetic and pharmacodynamic properties for in vivo applications. Finally, successful therapeutic application of RNAi will depend on the development of improved siRNA delivery strategies that combine high specificity and efficiency with a low immunostimulatory and tumorigenic potential.

Export and transport of tRNA are coupled to a multi-protein complex.

Vigilin is a ubiquitous multi heterogeneous nuclear ribonucleoprotein (hnRNP) K homologous (KH)-domain protein. Here we demonstrate that purified recombinant human vigilin binds tRNA molecules with high affinity, although with limited specificity. Nuclear microinjection experiments revealed for the first time that the immuno-affinity-purified nuclear vigilin core complex (VCC(N)) as well as recombinant vigilin accelerate tRNA export from the nucleus in human cells. The nuclear tRNA receptor exportin-t is part of the VCC(N). Elongation factor (EF)-1alpha is enriched in VCC(N) and its cytoplasmic counterpart VCC(C), whereas EF-1beta, EF-1gamma and EF-1delta are basically confined to the VCC(C). Our results suggest further that vigilin and exportin-t might interact during tRNA export, provide evidence that the channeled tRNA cycle is already initiated in the nucleus, and illustrate that intracellular tRNA trafficking is associated with discrete changes in the composition of cellular cytoplasmic multi-protein complexes containing tRNA.

Northern blot detection of endogenous small RNAs (∼14 nt) in bacterial total RNA extracts

Here we describe a northern blot procedure that allows the detection of endogenous RNAs as small as ∼14 nt in total RNA extracts from bacteria. RNAs that small and as part of total bacterial RNA extracts usually escape detection by northern blotting. The approach combines LNA probes 5′-digoxigenin-endlabeled for non-radioactive probe detection with 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide-mediated chemical crosslinking of RNAs to nylon membranes, and necessitates the use of native PAGE either with the TBE or MOPS buffer system.

Analysis of Transcriptional Regulation of the Human miR-17-92 Cluster; Evidence for Involvement of Pim-1

The human polycistronic miRNA cluster miR-17-92 is frequently overexpressed in hematopoietic malignancies and cancers. Its transcription is in part controlled by an E2F-regulated host gene promoter. An intronic A/T-rich region directly upstream of the miRNA coding region also contributes to cluster expression. Our deletion analysis of the A/T-rich region revealed a strong dependence on c-Myc binding to the functional E3 site. Yet, constructs lacking the 5′-proximal ~1.3 kb or 3′-distal ~0.1 kb of the 1.5 kb A/T-rich region still retained residual specific promoter activity, suggesting multiple transcription start sites (TSS) in this region. Furthermore, the protooncogenic kinase, Pim-1, its phosphorylation target HP1γ and c-Myc colocalize to the E3 region, as inferred from chromatin immunoprecipitation. Analysis of pri-miR-17-92 expression levels in K562 and HeLa cells revealed that silencing of E2F3, c-Myc or Pim-1 negatively affects cluster expression, with a synergistic effect caused by c-Myc/Pim-1 double knockdown in HeLa cells. Thus, we show, for the first time, that the protooncogene Pim-1 is part of the network that regulates transcription of the human miR-17-92 cluster.

RNA Binding of Ebola Virus VP30 Is Essential for Activating Viral Transcription

ABSTRACT The template for Ebola virus (EBOV) transcription and replication is the helical viral nucleocapsid composed of the viral negative-sense (−) RNA genome, which is complexed by the nucleoprotein (NP), VP35, polymerase L, VP24, and VP30. While viral replication is exerted by polymerase L and its cofactor VP35, EBOV mRNA synthesis is regulated by the viral nucleocapsid protein VP30, an essential EBOV-specific transcription factor. VP30 is a homohexameric phosphoprotein containing a nonconventional zinc finger. The transcriptional support activity of VP30 is strongly influenced by its phosphorylation state. We studied here how RNA binding contributed to VP30s function in transcriptional activation. Using a novel mobility shift assay and the 3′-terminal 154 nucleotides of the EBOV genome as a standard RNA substrate, we detected that RNA binding of VP30 was severely impaired by VP30 mutations that (i) destroy the proteins capability to form homohexamers, (ii) disrupt the integrity of its zinc finger domain, (iii) mimic its fully phosphorylated state, or (iv) alter the putative RNA binding region. RNA binding of the mutant VP30 proteins correlated strongly with their transcriptional support activity. Furthermore, we showed that the interaction between VP30 and the polymerase cofactor VP35 is RNA dependent, while formation of VP30 homohexamers and VP35 homotetramers is not. Our data indicate that RNA binding of VP30 is essential for its transcriptional support activity and stabilizes complexes of VP35/L polymerase with the (−) RNA template to favor productive transcriptional initiation in the presence of termination-active RNA secondary structures. IMPORTANCE Ebola virus causes severe fevers with unusually high case fatality rates. The recent outbreak of Ebola virus in West Africa claimed more than 11,000 lives and threatened to destabilize a whole region because of its dramatic effects on the public health systems. It is currently not completely understood how Ebola virus manages to balance viral transcription and replication in the infected cells. This study shows that transcriptional support activity of the Ebola virus transcription factor VP30 is highly correlated with its ability to bind viral RNA. The interaction between VP30 and VP35, the Ebola virus polymerase cofactor, is dependent on the presence of RNA as well. Our data contribute to the understanding of the dynamic interplay between nucleocapsid proteins and the viral RNA template in order to promote viral RNA synthesis.

The natural compound silvestrol is a potent inhibitor of Ebola virus replication.

&NA; The DEAD‐box RNA helicase eIF4A, which is part of the heterotrimeric translation initiation complex in eukaryotes, is an important novel drug target in cancer research because its helicase activity is required to unwind extended and highly structured 5′‐UTRs of several proto‐oncogenes. Silvestrol, a natural compound isolated from the plant Aglaia foveolata, is a highly efficient, non‐toxic and specific inhibitor of eIF4A. Importantly, 5′‐capped viral mRNAs often contain structured 5′‐UTRs as well, which may suggest a dependence on eIF4A for their translation by the host protein synthesis machinery. In view of the recent Ebola virus (EBOV) outbreak in West Africa, the identification of potent antiviral compounds is urgently required. Since Ebola mRNAs are 5′‐capped and harbor RNA secondary structures in their extended 5′‐UTRs, we initiated a BSL4 study to analyze silvestrol in EBOV‐infected Huh‐7 cells and in primary human macrophages for its antiviral activity. We observed that silvestrol inhibits EBOV infection at low nanomolar concentrations, as inferred from large reductions of viral titers. This correlated with an almost complete disappearance of EBOV proteins, comparable in effect to the translational shutdown of expression of the proto‐oncoprotein PIM1, a cellular kinase known to be affected by silvestrol. Effective silvestrol concentrations were non‐toxic in the tested cell systems. Thus, silvestrol appears to be a promising first‐line drug for the treatment of acute EBOV and possibly other viral infections. Graphical abstract: Figure. No caption available. HighlightsThe rocaglate silvestrol, a specific eIF4A helicase inhibitor, is a potent antiviral compound to treat Ebola infections.Silvestrol inhibits EBOV in human primary macrophages at low nanomolar concentrations.Silvestrol is not toxic in vitro and in vivo at concentrations of effective antiviral activity.Inhibition of cap‐dependent translation initiation is a potent antiviral strategy.

RNA binding specificity of Ebola virus transcription factor VP30

ABSTRACT The transcription factor VP30 of the non-segmented RNA negative strand Ebola virus balances viral transcription and replication. Here, we comprehensively studied RNA binding by VP30. Using a novel VP30:RNA electrophoretic mobility shift assay, we tested truncated variants of 2 potential natural RNA substrates of VP30 - the genomic Ebola viral 3′-leader region and its complementary antigenomic counterpart (each ∼155 nt in length) - and a series of other non-viral RNAs. Based on oligonucleotide interference, the major VP30 binding region on the genomic 3′-leader substrate was assigned to the internal expanded single-stranded region (∼ nt 125–80). Best binding to VP30 was obtained with ssRNAs of optimally ∼ 40 nt and mixed base composition; underrepresentation of purines or pyrimidines was tolerated, but homopolymeric sequences impaired binding. A stem-loop structure, particularly at the 3′-end or positioned internally, supports stable binding to VP30. In contrast, dsRNA or RNAs exposing large internal loops flanked by entirely helical arms on both sides are not bound. Introduction of a 5´-Cap(0) structure impaired VP30 binding. Also, ssDNAs bind substantially weaker than isosequential ssRNAs and heparin competes with RNA for binding to VP30, indicating that ribose 2′-hydroxyls and electrostatic contacts of the phosphate groups contribute to the formation of VP30:RNA complexes. Our results indicate a rather relaxed RNA binding specificity of filoviral VP30, which largely differs from that of the functionally related transcription factor of the Paramyxoviridae which binds to ssRNAs as short as 13 nt with a preference for oligo(A) sequences.

PEI-complexed LNA antiseeds as miRNA inhibitors

Antisense inhibition of oncogenic or other disease-related miRNAs and miRNA families in vivo may provide novel therapeutic strategies. However, this approach relies on the development of potent miRNA inhibitors and their efficient delivery into cells. Here, we introduce short seed-directed LNA oligonucleotides (12- or 14-mer antiseeds) with a phosphodiester backbone (PO) for efficient miRNA inhibition. We have analyzed such LNA (PO) antiseeds using a let-7a-controlled luciferase reporter assay and identified them as active miRNA inhibitors in vitro. Moreover, LNA (PO) 14-mer antiseeds against ongogenic miR-17–5p and miR-20a derepress endogenous p21 expression more persistently than corresponding miRNA hairpin inhibitors, which are often used to inhibit miRNA function. Further analysis of the antiseed-mediated derepression of p21 in luciferase reporter constructs - containing the 3′-UTR of p21 and harboring two binding sites for miRNAs of the miR-106b family - provided evidence that the LNA antiseeds inhibit miRNA families while hairpin inhibitors act in a miRNA-specific manner. The derepression caused by LNA antiseeds is specific, as demonstrated via seed mutagenesis of the miR-106b target sites. Importantly, we show functional delivery of LNA (PO) 14-mer antiseeds into cells upon complexation with polyethylenimine (PEI F25-LMW), which leads to the formation of polymeric nanoparticles. In contrast, attempts to deliver a functional seed-directed tiny LNA 8-mer with a phosphorothioate backbone (PS) by formulation with PEI F25-LMW remained unsuccessful. In conclusion, LNA (PO) 14-mer antiseeds are attractive miRNA inhibitors, and their PEI-based delivery may represent a promising new strategy for therapeutic applications.