Discovery of small molecules that target a tertiary-structured RNA.

There is growing interest in therapeutic intervention that targets disease-relevant RNAs using small molecules. While there have been some successes in RNA-targeted small-molecule discovery, a deeper understanding of structure-activity relationships in pursuing these targets has remained elusive. One of the best-studied tertiary-structured RNAs is the theophylline aptamer, which binds theophylline with high affinity and selectivity. Although not a drug target, this aptamer has had many applications, especially pertaining to genetic control circuits. Heretofore, no compound has been shown to bind the theophylline aptamer with greater affinity than theophylline itself. However, by carrying out a high-throughput screen of low-molecular-weight compounds, several unique hits were identified that are chemically distinct from theophylline and bind with up to 340-fold greater affinity. Multiple atomic-resolution X-ray crystal structures were determined to investigate the binding mode of theophylline and four of the best hits. These structures reveal both the rigidity of the theophylline aptamer binding pocket and the opportunity for other ligands to bind more tightly in this pocket by forming additional hydrogen-bonding interactions. These results give encouragement that the same approaches to drug discovery that have been applied so successfully to proteins can also be applied to RNAs.

3D Columnar Phase of Stacked Short DNA Organized by Coherent Membrane Undulations.

We report on the discovery of a new organized lipid-nucleic acid phase upon intercalation of blunt duplexes of short DNA (sDNA) within cationic multilayer fluid membranes. End-to-end interactions between sDNA leads to columnar stacks. At high membrane charge density, with the inter-sDNA column spacing (dsDNA) comparable but larger than the diameter of sDNA, a 2D columnar phase (i.e., a 2D smectic) is found similar to the phase in cationic liposome-DNA complexes with long lambda-phage DNA. Remarkably, with increasing dsDNA as the membrane charge density is lowered, a transition is observed to a 3D columnar phase of stacked sDNA. This occurs even though direct DNA-DNA electrostatic interactions across layers are screened by diffusing cationic lipids near the phosphate groups of sDNA. Softening of the membrane bending rigidity (κ), which further promotes membrane undulations, significantly enhances the 3D columnar phase. These observations are consistent with a model by Schiessel and Aranda-Espinoza where local membrane undulations, due to electrostatically induced membrane wrapping around sDNA columns, phase lock from layer-to-layer, thereby precipitating coherent "crystal-like" undulations coupled to sDNA columns with long-range position and orientation order. The finding that this new phase is stable at large dsDNA and enhanced with decreasing κ is further supportive of the model where the elastic cost of membrane deformation per unit area around sDNA columns (∝ κh2/dsDNA4, h2 = sum of square of amplitudes of the inner and outer monolayer undulations) is strongly reduced relative to the favorable electrostatic attractions of partially wrapped membrane around sDNA columns. The findings have broad implications in the design of membrane-mediated assembly of functional nanoparticles in 3D.

TLR3 signaling is downregulated by a MAVS isoform in epithelial cells.

Innate immune responses to dsRNA result in signaling through the TLR3 pathway and/or the RIG-I/MDA-5/MAVS pathway which can activate type I IFN, proinflammatory cytokines and apoptosis. It is not clear whether MAVS could play a role in TLR3-dependent responses to extracellular dsRNA. Using a model of epithelial cells that express a functional TLR3 signaling pathway, we found that TLR3-dependent responses to extracellular dsRNA are negatively regulated by MAVS, precisely "miniMAVS", a recently described 50kDa isoform of MAVS. This regulation of TLR3 by a MAVS isoform constitutes an endogenous regulatory mechanism in epithelial cells that could help prevent a potentially damaging excessive inflammatory response.

GM-CSF produced by nonhematopoietic cells is required for early epithelial cell proliferation and repair of injured colonic mucosa.

GM-CSF is a growth factor that promotes the survival and activation of macrophages and granulocytes, as well as dendritic cell differentiation and survival in vitro. The mechanism by which exogenous GM-CSF ameliorates the severity of Crohn's disease in humans and colitis in murine models has mainly been considered to reflect its activity on myeloid cells. We used GM-CSF-deficient (GM-CSF(-/-)) mice to probe the functional role of endogenous host-produced GM-CSF in a colitis model induced after injury to the colon epithelium. Dextran sodium sulfate (DSS), at doses that resulted in little epithelial damage and mucosal ulceration in wild type mice, caused marked colon ulceration and delayed ulcer healing in GM-CSF(-/-) mice. Colon crypt epithelial cell proliferation in vivo was significantly decreased in GM-CSF(-/-) mice at early times after DSS injury. This was paralleled by decreased expression of crypt epithelial cell genes involved in cell cycle, proliferation, and wound healing. Decreased crypt cell proliferation and delayed ulcer healing in GM-CSF(-/-) mice were rescued by exogenous GM-CSF, indicating the lack of a developmental abnormality in the epithelial cell proliferative response in those mice. Nonhematopoietic cells, and not myeloid cells, produced the GM-CSF important for colon epithelial proliferation after DSS-induced injury, as revealed by bone marrow chimera and dendritic cell-depletion experiments, with colon epithelial cells being the cellular source of GM-CSF. Endogenous epithelial cell-produced GM-CSF has a novel nonredundant role in facilitating epithelial cell proliferation and ulcer healing in response to injury of the colon crypt epithelium.

TLR3, TRIF, and caspase 8 determine double-stranded RNA-induced epithelial cell death and survival in vivo.

TLR3 signaling is activated by dsRNA, a virus-associated molecular pattern. Injection of dsRNA into mice induced a rapid, dramatic, and reversible remodeling of the small intestinal mucosa with significant villus shortening. Villus shortening was preceded by increased caspase 3 and 8 activation and apoptosis of intestinal epithelial cells (IECs) located in the mid to upper villus with ensuing luminal fluid accumulation and diarrhea because of an increased secretory state. Mice lacking TLR3 or the adaptor molelcule TRIF mice were completely protected from dsRNA-induced IEC apoptosis, villus shortening, and diarrhea. dsRNA-induced apoptosis was independent of TNF signaling. Notably, NF-κB signaling through IκB kinase ß protected crypt IECs but did not protect villus IECs from dsRNA-induced or TNF-induced apoptosis. dsRNA did not induce early caspase 3 activation with subsequent villus shortening in mice lacking caspase 8 in IECs but instead caused villus destruction with a loss of small intestinal surface epithelium and death. Consistent with direct activation of the TLR3-TRIF-caspase 8 signaling pathway by dsRNA in IECs, dsRNA-induced signaling of apoptosis was independent of non-TLR3 dsRNA signaling pathways, IL-15, TNF, IL-1, IL-6, IFN regulatory factor 3, type I IFN receptor, adaptive immunity, as well as dendritic cells, NK cells, and other hematopoietic cells. We conclude that dsRNA activation of the TLR3-TRIF-caspase 8 signaling pathway in IECs has a significant impact on the structure and function of the small intestinal mucosa and suggest signaling through this pathway has a host protective role during infection with viral pathogens.

Protein kinase PKR amplification of interferon ß induction occurs through initiation factor eIF-2α-mediated translational control.

The protein kinase PKR is activated by RNA with double-stranded (ds) structure and subsequently impairs translation through phosphorylation of protein synthesis initiation factor eIF-2α. PKR also mediates activation of signal transduction pathways leading to interferon beta (IFN-ß) gene induction following virus-infection or RNA transfection. We previously demonstrated in measles virus-infected cells that PKR is required for the maximal induction of IFN-ß gene expression by the interferon promoter stimulator gene 1 (IPS-1) adaptor-dependent cytosolic RNA sensor pathway. While both IPS-1 and PKR are important mediators of IFN-ß induction, with PKR contributing to an enhanced NF-κB activation, the mechanism by which PKR enhances NF-κB activity and amplifies IFN-ß induction is unresolved. Herein we tested the possibility that PKR could activate signal transduction pathways indirectly through translational control responses. Following transfection with synthetic or natural dsRNAs or infection with measles virus, we observed increased mRNA but decreased protein levels for the inhibitor of NF-κB signaling, IκB-α, that correlated with PKR activation and eIF-2α phosphorylation. Importantly, knockdown of PKR increased IκB-α protein levels and impaired IFN-ß induction. Additionally, inhibition of translation by cycloheximide treatment rescued IFN-ß induction following PKR knockdown but not IPS-1 knockdown. Mutation of eIF-2α to prevent phosphorylation also impaired IFN-ß induction in PKR-sufficient virus-infected cells. These results suggest that an eIF-2α-dependent translation inhibition mechanism is sufficient to explain the PKR-mediated amplification of IPS-1-dependent IFN-ß induction by foreign RNA.

RIG-I/MDA5/MAVS are required to signal a protective IFN response in rotavirus-infected intestinal epithelium.

Rotavirus is a dsRNA virus that infects epithelial cells that line the surface of the small intestine. It causes severe diarrheal illness in children and ∼500,000 deaths per year worldwide. We studied the mechanisms by which intestinal epithelial cells (IECs) sense rotavirus infection and signal IFN-ß production, and investigated the importance of IFN-ß production by IECs for controlling rotavirus production by intestinal epithelium and virus excretion in the feces. In contrast with most RNA viruses, which interact with either retinoic acid-inducible gene I (RIG-I) or melanoma differentiation-associated gene 5 (MDA5) inside cells, rotavirus was sensed by both RIG-I and MDA5, alone and in combination. Rotavirus did not signal IFN-ß through either of the dsRNA sensors TLR3 or dsRNA-activated protein kinase (PKR). Silencing RIG-I or MDA5, or their common adaptor protein mitochondrial antiviral signaling protein (MAVS), significantly decreased IFN-ß production and increased rotavirus titers in infected IECs. Overexpression of laboratory of genetics and physiology 2, a RIG-I-like receptor that interacts with viral RNA but lacks the caspase activation and recruitment domains required for signaling through MAVS, significantly decreased IFN-ß production and increased rotavirus titers in infected IECs. Rotavirus-infected mice lacking MAVS, but not those lacking TLR3, TRIF, or PKR, produced significantly less IFN-ß and increased amounts of virus in the intestinal epithelium, and shed increased quantities of virus in the feces. We conclude that RIG-I or MDA5 signaling through MAVS is required for the activation of IFN-ß production by rotavirus-infected IECs and has a functionally important role in determining the magnitude of rotavirus replication in the intestinal epithelium.

Two-dimensional packing of short DNA with nonpairing overhangs in cationic liposome-DNA complexes: from Onsager nematics to columnar nematics with finite-length columns.

We report the formation of liquid crystalline (LC) phases of short double-stranded DNA with nonpairing (nonsticky) overhangs, confined between two-dimensional (2D) lipid bilayers of cationic liposome-DNA complexes. In a landmark study (Science2007, 318, 1276), Nakata et al. reported on the discovery of strong end-to-end stacking interactions between short DNAs (sDNAs) with blunt ends, leading to the formation of 3D nematic (N) and columnar LC phases. Employing synchrotron small-angle X-ray scattering, we have studied the interplay between shape anisotropy-induced and DNA end-to-end interaction-induced N ordering for 11, 24, and 48 bp sDNA rods with single-stranded oligo-thymine (T) overhangs modulating the end-to-end interactions. For suppressed stacking interactions with 10-T overhangs, the volume fraction of sDNA at which the 2D isotropic (I)-to-N transition occurs for 24 and 48 bp sDNA rods depended on their length-to-width (L/D) shape anisotropy, qualitatively consistent with Onsager's theory for the entropic alignment of rigid rods. As the overhang length is reduced from 10 to 5 and 2 T for 24 and 48 bp sDNA, the N-to-I transition occurs at lower volume fractions, indicating the onset of some degree of end-to-end stacking interactions. The 11 bp sDNA rods with 5- and 10-T overhangs remain in the I phase, consistent with their small shape anisotropy (L/D ≈ 1.9) below the limit for Onsager LC ordering. Unexpectedly, in contrast to the behavior of 24 and 48 bp sDNA, the end-to-end interactions between 11 bp sDNA rods with 2-T overhangs set in dramatically, and a novel 2D columnar N phase (N(C)) with finite-length columns formed. The building blocks of this phase are comprised of 1D stacks of (on average) four 11 bp DNA-2T rods with an effective L(stacked)/D ≈ 8.2. Our findings have implications for the DNA-directed assembly of nanoparticles on 2D platforms via end-to-end interactions and in designing optimally packed LC phases of short anisotropic biomolecules (such as peptides and short-interfering RNAs) on nanoparticle membranes, which are used in gene silencing and chemical delivery.

Mechanisms of protein kinase PKR-mediated amplification of beta interferon induction by C protein-deficient measles virus.

The measles virus P gene products V and C antagonize the host interferon (IFN) response, blocking both IFN signaling and production. Using Moraten vaccine strain-derived measles virus and isogenic mutants deficient for either V or C protein production (V(ko) and C(ko), respectively), we observed that the C(ko) virus was a potent inducer of IFN-beta, while induction by V(ko) virus was an order of magnitude lower than that by the C(ko) virus. The parental recombinant Moraten virus did not significantly induce IFN-beta. The enhanced IFN-inducing capacity of the C(ko) virus correlated with an enhanced activation of IFN regulatory factor 3 (IRF-3), NF-kappaB, and ATF-2 in C(ko)-infected compared to V(ko) or parental virus-infected cells. Furthermore, protein kinase PKR and mitochondrial adapter IPS-1 were required for maximal C(ko)-mediated IFN-beta induction, which correlated with the PKR-mediated enhancement of mitogen-activated protein kinase and NF-kappaB activation. Our results reveal multiple consequences of C protein expression and document an important function for PKR as an enhancer of IFN-beta induction during measles virus infection.

Cationic liposome-nucleic acid complexes for gene delivery and silencing: pathways and mechanisms for plasmid DNA and siRNA.

Motivated by the promises of gene therapy, there is great interest in developing non-viral lipid-based vectors for therapeutic applications due to their low immunogenicity, low toxicity, ease of production, and the potential of transferring large pieces of DNA into cells. In fact, cationic liposome (CL) based vectors are among the prevalent synthetic carriers of nucleic acids (NAs) currently used in gene therapy clinical trials worldwide. These vectors are studied both for gene delivery with CL-DNA complexes and gene silencing with CL-siRNA (short interfering RNA) complexes. However, their transfection efficiencies and silencing efficiencies remain low compared to those of engineered viral vectors. This reflects the currently poor understanding of transfection-related mechanisms at the molecular and self-assembled levels, including a lack of knowledge about interactions between membranes and double stranded NAs and between CL-NA complexes and cellular components. In this review we describe our recent efforts to improve the mechanistic understanding of transfection by CL-NA complexes, which will help to design optimal lipid-based carriers of DNA and siRNA for therapeutic gene delivery and gene silencing.

Dexamethasone inhibits respiratory syncytial virus-driven mucus production while increasing viral replication without altering antiviral interferon signaling.

Respiratory syncytial virus (RSV) infection can cause mucus overproduction and bronchiolitis in infants leading to severe disease and hospitalization. As a therapeutic strategy, immune modulatory agents may help prevent RSV-driven immune responses that cause severe airway disease. We developed a high throughput screen to identify compounds that reduced RSV-driven mucin 5AC (Muc5AC) expression and identified dexamethasone. Despite leading to a pronounced reduction in RSV-driven Muc5AC, dexamethasone increased RSV infection in vitro and delayed viral clearance in mice. This correlated with reduced expression of a subset of immune response genes and reduced lymphocyte infiltration in vivo. Interestingly, dexamethasone increased RSV infection levels without altering antiviral interferon signaling. In summary, the immunosuppressive activities of dexamethasone had favorable inhibitory effects on RSV-driven mucus production yet prevented immune defense activities that limit RSV infection in vitro and in vivo. These findings offer an explanation for the lack of efficacy of glucocorticoids in RSV-infected patients.

The immunopathogenesis of celiac disease reveals possible therapies beyond the gluten-free diet.

Celiac disease is a T cell-mediated autoimmune inflammatory disease of the small intestine that is activated by gluten. The diagnosis of celiac disease is challenging as patients display a wide range of symptoms and some are asymptomatic. A lifelong gluten-free diet is the only currently approved treatment of celiac disease. Although the diet is safe and effective, the compliance rates and patient acceptance vary. Furthermore, many patients treated with a gluten-free diet continue to be mildly to severely symptomatic with persistent histological abnormalities, and a small number of patients develop refractory celiac disease. New therapeutic adjuncts and potential alternatives to the gluten-free diet could improve the treatment options for these patients. Advances in understanding the immunopathogenesis of celiac disease have suggested several types of therapeutic strategies that may augment or supplant the gluten-free diet. Some of these strategies attempt to decrease the immunogenicity of gluten-containing grains by manipulating the grain itself or by using oral enzymes to break down immunogenic peptides that normally remain intact during digestion. Other strategies focus on preventing the absorption of these peptides, preventing tissue transglutaminase from rendering gluten peptides more immunogenic, or inhibiting their binding to celiac disease-specific antigen-presenting molecules. Strategies that limit T cell migration to the small intestine or that reestablish mucosal homeostasis and tolerance to gluten antigens are also being explored. Additionally, it is vital to develop new therapeutic options for refractory celiac disease patients. This review highlights therapeutic strategies that may ultimately improve the health and well-being of individuals with celiac disease.

The RNA-activated protein kinase enhances the induction of interferon-beta and apoptosis mediated by cytoplasmic RNA sensors.

Detection of foreign RNA by the innate immune system can trigger the induction of type I interferon (IFN) and apoptosis. Important antiviral defense pathways that result in type I IFN production following the recognition of foreign double-stranded RNA (dsRNA) include the RIG-I family helicases and IPS-1 adaptor cytosolic pathway and the Toll-like receptor 3 and TIR domain-containing adaptor-inducing IFN-beta (TRIF) adaptor membrane-associated pathway, both of which activate IFN regulatory factor 3 (IRF3). In addition to triggering an innate immune response, dsRNAs are widely used to mediate gene-selective silencing in mammalian cells by the RNA interference pathway. We investigated the ability of short interfering RNAs, including T7 phage polymerase-synthesized RNA (PRNA), which like some viral RNAs contains a 5'-triphosphate, to selectively silence gene expression and to cause induction of IFN-beta and apoptosis. We found that PRNA-mediated gene silencing and associated nonspecific pro-apoptotic and IFN-inducing effects were dependent on the cell line and RNA length. Double-stranded PRNAs 50 nucleotides long as well as polyinosinic-polycytidylic acid activated the RNA-dependent protein kinase (PKR) and induced significant levels of IFN-beta and apoptosis, whereas shorter PRNAs and chemically synthesized dsRNAs did not. Effector caspase activation and apoptosis following RNA transfection was enhanced by pretreatment with IFN, and removal of the 5'-phosphate from PRNAs decreased induction of both IFN-beta and apoptosis. PKR, in addition to IPS-1 and IRF3 but not TRIF, was required for maximal type I IFN-beta induction and the induction of apoptosis by both transfected PRNAs and polyinosinic-polycytidylic acid.

Structure and gene silencing activities of monovalent and pentavalent cationic lipid vectors complexed with siRNA.

Small interfering RNAs (siRNAs) of 19-25 bp mediate the cleavage of complementary mRNA, leading to post-transcriptional gene silencing. We examined cationic lipid (CL)-mediated delivery of siRNA into mammalian cells and made comparisons to CL-based DNA delivery. The effect of lipid composition and headgroup charge on the biophysical and biological properties of CL-siRNA vectors was determined. X-ray diffraction revealed that CL-siRNA complexes exhibited lamellar and inverted hexagonal phases, qualitatively similar to CL-DNA complexes, but also formed other nonlamellar structures. Surprisingly, optimally formulated inverted hexagonal 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP)/1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine (DOPE) CL-siRNA complexes exhibited high toxicity and much lower target-specific gene silencing than lamellar CL-siRNA complexes even though optimally formulated, inverted hexagonal CL-DNA complexes show high transfection efficiency in cell culture. We further found that efficient silencing required cationic lipid/nucleic acid molar charge ratios (rhochg) nearly an order of magnitude larger than those yielding efficiently transfecting CL-DNA complexes. This second unexpected finding has implications for cell toxicity. Multivalent lipids (MVLs) require a smaller number of cationic lipids at a given rhochg of the complex. Consistent with this observation, the pentavalent lipid MVL5 exhibited lower toxicity and superior silencing efficiency over a large range in both the lipid composition and rhochg when compared to monovalent DOTAP. Most importantly, MVL5 achieved much higher total knockdown of the target gene in CL-siRNA complex regimes where toxicity was low. This property of CL-siRNA complexes contrasts to CL-DNA complexes, where the optimized transfection efficiencies of multivalent and monovalent lipids are comparable.