GOLGA8 increases bulk antisense oligonucleotide uptake and activity in mammalian cells.

Antisense oligonucleotides (ASOs) are short synthetic nucleic acids that recognize and bind to complementary RNA to modulate gene expression. It is well established that single-stranded, phosphorothioate-modified ASOs enter cells independent of carrier molecules, primarily via endocytic pathways, but that only a small portion of internalized ASO is released into the cytosol and/or nucleus, rendering the majority of ASO inaccessible to the targeted RNA. Identifying pathways that can increase the available ASO pool is valuable as a research tool and therapeutically. Here, we conducted a functional genomic screen for ASO activity by engineering GFP splice reporter cells and applying genome-wide CRISPR gene activation. The screen can identify factors that enhance ASO splice modulation activity. Characterization of hit genes uncovered GOLGA8, a largely uncharacterized protein, as a novel positive regulator enhancing ASO activity by ∼2-fold. Bulk ASO uptake is 2- to 5-fold higher in GOLGA8-overexpressing cells where GOLGA8 and ASOs are observed in the same intracellular compartments. We find GOLGA8 is highly localized to the trans-Golgi and readily detectable at the plasma membrane. Interestingly, overexpression of GOLGA8 increased activity for both splice modulation and RNase H1-dependent ASOs. Taken together, these results support a novel role for GOLGA8 in productive ASO uptake.

Steric Inhibition of 5' UTR Regulatory Elements Results in Upregulation of Human CFTR.

Cystic fibrosis (CF) is an autosomal recessive monogenic disease caused by mutations in the CFTR gene. Therapeutic approaches that are focused on correcting CFTR protein face the challenge of the heterogeneity in CFTR mutations and resulting defects. Thus, while several small molecules directed at CFTR show benefit in the clinic for subsets of CF patients, these drugs cannot treat all CF patients. Additionally, the clinical benefit from treatment with these modulators could be enhanced with novel therapies. To address this unmet need, we utilized an approach to increase CFTR protein levels through antisense oligonucleotide (ASO)-mediated steric inhibition of 5' UTR regulatory elements. We identified ASOs to upregulate CFTR protein expression and confirmed the regulatory role of the sites amenable to ASO-mediated upregulation. Two ASOs were investigated further, and both increased CFTR protein expression and function in cell lines and primary human bronchial epithelial cells with distinct CF genotypes. ASO treatment further increased CFTR function in almost all CF genotypes tested on top of treatment with the FDA approved drug Symdeko (ivacaftor and tezacaftor). Thus, we present a novel approach to CFTR therapeutic intervention, through ASO-mediated modulation of translation.

Oncogenic Role of THOR, a Conserved Cancer/Testis Long Non-coding RNA.

Large-scale transcriptome sequencing efforts have vastly expanded the catalog of long non-coding RNAs (lncRNAs) with varying evolutionary conservation, lineage expression, and cancer specificity. Here, we functionally characterize a novel ultraconserved lncRNA, THOR (ENSG00000226856), which exhibits expression exclusively in testis and a broad range of human cancers. THOR knockdown and overexpression in multiple cell lines and animal models alters cell or tumor growth supporting an oncogenic role. We discovered a conserved interaction of THOR with IGF2BP1 and show that THOR contributes to the mRNA stabilization activities of IGF2BP1. Notably, transgenic THOR knockout produced fertilization defects in zebrafish and also conferred a resistance to melanoma onset. Likewise, ectopic expression of human THOR in zebrafish accelerated the onset of melanoma. THOR represents a novel class of functionally important cancer/testis lncRNAs whose structure and function have undergone positive evolutionary selection.

Antisense oligonucleotides targeting translation inhibitory elements in 5' UTRs can selectively increase protein levels.

A variety of diseases are caused by deficiencies in amounts or activity of key proteins. An approach that increases the amount of a specific protein might be of therapeutic benefit. We reasoned that translation could be specifically enhanced using trans-acting agents that counter the function of negative regulatory elements present in the 5' UTRs of some mRNAs. We recently showed that translation can be enhanced by antisense oligonucleotides (ASOs) that target upstream open reading frames. Here we report the amount of a protein can also be selectively increased using ASOs designed to hybridize to other translation inhibitory elements in 5' UTRs. Levels of human RNASEH1, LDLR, and ACP1 and of mouse ACP1 and ARF1 were increased up to 2.7-fold in different cell types and species upon treatment with chemically modified ASOs targeting 5' UTR inhibitory regions in the mRNAs encoding these proteins. The activities of ASOs in enhancing translation were sequence and position dependent and required helicase activity. The ASOs appear to improve the recruitment of translation initiation factors to the target mRNA. Importantly, ASOs targeting ACP1 mRNA significantly increased the level of ACP1 protein in mice, suggesting that this approach has therapeutic and research potentials.

Identification and Validation of PCAT14 as Prognostic Biomarker in Prostate Cancer.

Rapid advances in the discovery of long noncoding RNAs (lncRNAs) have identified lineage- and cancer-specific biomarkers that may be relevant in the clinical management of prostate cancer (PCa). Here we assembled and analyzed a large RNA-seq dataset, from 585 patient samples, including benign prostate tissue and both localized and metastatic PCa to discover and validate differentially expressed genes associated with disease aggressiveness. We performed Sample Set Enrichment Analysis (SSEA) and identified genes associated with low versus high Gleason score in the RNA-seq database. Comparing Gleason 6 versus 9+ PCa samples, we identified 99 differentially expressed genes with variable association to Gleason grade as well as robust expression in prostate cancer. The top-ranked novel lncRNA PCAT14, exhibits both cancer and lineage specificity. On multivariate analysis, low PCAT14 expression independently predicts for BPFS (P=.00126), PSS (P=.0385), and MFS (P=.000609), with trends for OS as well (P=.056). An RNA in-situ hybridization (ISH) assay for PCAT14 distinguished benign vs malignant cases, as well as high vs low Gleason disease. PCAT14 is transcriptionally regulated by AR, and endogenous PCAT14 overexpression suppresses cell invasion. Thus, Using RNA-sequencing data we identify PCAT14, a novel prostate cancer and lineage-specific lncRNA. PCAT14 is highly expressed in low grade disease and loss of PCAT14 predicts for disease aggressiveness and recurrence.

Integrated Safety Assessment of 2'-O-Methoxyethyl Chimeric Antisense Oligonucleotides in NonHuman Primates and Healthy Human Volunteers.

The common chemical and biological properties of antisense oligonucleotides provide the opportunity to identify and characterize chemical class effects across species. The chemical class that has proven to be the most versatile and best characterized is the 2'-O-methoxyethyl chimeric antisense oligonucleotides. In this report we present an integrated safety assessment of data obtained from controlled dose-ranging studies in nonhuman primates (macaques) and healthy human volunteers for 12 unique 2'-O-methoxyethyl chimeric antisense oligonucleotides. Safety was assessed by the incidence of safety signals in standardized laboratory tests for kidney and liver function, hematology, and complement activation; as well as by the mean test results as a function of dose level over time. At high doses a number of toxicities were observed in nonhuman primates. However, no class safety effects were identified in healthy human volunteers from this integrated data analysis. Effects on complement in nonhuman primates were not observed in humans. Nonhuman primates predicted safe doses in humans, but over predicted risk of complement activation and effects on platelets. Although limited to a single chemical class, comparisons from this analysis are considered valid and accurate based on the carefully controlled setting for the specified study populations and within the total exposures studied.

Hepatotoxicity of high affinity gapmer antisense oligonucleotides is mediated by RNase H1 dependent promiscuous reduction of very long pre-mRNA transcripts.

High affinity antisense oligonucleotides (ASOs) containing bicylic modifications (BNA) such as locked nucleic acid (LNA) designed to induce target RNA cleavage have been shown to have enhanced potency along with a higher propensity to cause hepatotoxicity. In order to understand the mechanism of this hepatotoxicity, transcriptional profiles were collected from the livers of mice treated with a panel of highly efficacious hepatotoxic or non-hepatotoxic LNA ASOs. We observed highly selective transcript knockdown in mice treated with non-hepatotoxic LNA ASOs, while the levels of many unintended transcripts were reduced in mice treated with hepatotoxic LNA ASOs. This transcriptional signature was concurrent with on-target RNA reduction and preceded transaminitis. Remarkably, the mRNA transcripts commonly reduced by toxic LNA ASOs were generally not strongly associated with any particular biological process, cellular component or functional group. However, they tended to have much longer pre-mRNA transcripts. We also demonstrate that the off-target RNA knockdown and hepatotoxicity is attenuated by RNase H1 knockdown, and that this effect can be generalized to high affinity modifications beyond LNA. This suggests that for a certain set of ASOs containing high affinity modifications such as LNA, hepatotoxicity can occur as a result of unintended off-target RNase H1 dependent RNA degradation.

Targeting of repeated sequences unique to a gene results in significant increases in antisense oligonucleotide potency.

A new strategy for identifying potent RNase H-dependent antisense oligonucleotides (ASOs) is presented. Our analysis of the human transcriptome revealed that a significant proportion of genes contain unique repeated sequences of 16 or more nucleotides in length. Activities of ASOs targeting these repeated sites in several representative genes were compared to those of ASOs targeting unique single sites in the same transcript. Antisense activity at repeated sites was also evaluated in a highly controlled minigene system. Targeting both native and minigene repeat sites resulted in significant increases in potency as compared to targeting of non-repeated sites. The increased potency at these sites is a result of increased frequency of ASO/RNA interactions which, in turn, increases the probability of a productive interaction between the ASO/RNA heteroduplex and human RNase H1 in the cell. These results suggest a new, highly efficient strategy for rapid identification of highly potent ASOs.

Identification of REST-regulated genes and pathways using a REST-targeted antisense approach.

The repressor element-1 silencing transcription factor/neuron-restrictive silencer factor (REST/NRSF) is one of the first negative-acting transcriptional regulators implicated in vertebrate development thought to regulate hundreds of neuron-specific genes. However, its function in the adult system remains elusive. Here we employ second-generation antisense oligonucleotides (ASOs) to study the impact of rest-mediated suppression on gene expression. We demonstrate specific reductions in REST levels in vitro, and in vivo in mouse liver following treatment with ASOs, and we show that ASO mediated-REST suppression results in the elevation in expression of many neuronal genes including brain-derived neurotrophic factor, Synapsin1 (syn1) and ß3-tubulin in BALB/c liver. Furthermore, we show the elevation of the affected proteins in plasma following ASO treatment. Finally, microarray analysis was applied to identify a broad range of genes modulated by REST suppression in mouse liver. Our findings suggest that REST may be an important target for neurodegenerative diseases like Huntington's disease, is also involved in the regulation of a broad range of additional cellular pathways, and that the antisense approach is a viable strategy for selectively modulating REST activity in vivo.

Modeling of the water network at protein-RNA interfaces.

Water plays an important role in the mediation of biomolecular interactions. Thus, accurate prediction and evaluation of water-mediated interactions is an important element in the computational design of interfaces involving proteins, RNA, and DNA. Here, we use an algorithm (WATGEN) to predict the locations of interfacial water molecules for a data set of 224 protein-RNA interfaces. The accuracy of the prediction is validated against water molecules present in the X-ray structures of 105 of these complexes. The complexity of the water networks is deconvoluted through definition of the characteristics of each water molecule based on its bridging properties between the protein and RNA and on its depth in the interface with respect to the bulk solvent. This approach has the potential for scoring the water network for incorporation into the computational design of protein-RNA complexes.

Polyfunctional CD4+ T cell responses to a set of pathogenic arenaviruses provide broad population coverage.

BACKGROUND: Several arenaviruses cause severe hemorrhagic fever and aseptic meningitis in humans for which no licensed vaccines are available. A major obstacle for vaccine development is pathogen heterogeneity within the Arenaviridae family. Evidence in animal models and humans indicate that T cell and antibody-mediated immunity play important roles in controlling arenavirus infection and replication. Because CD4+ T cells are needed for optimal CD8+ T cell responses and to provide cognate help for B cells, knowledge of epitopes recognized by CD4+ T cells is critical to the development of an effective vaccine strategy against arenaviruses. Thus, the goal of the present study was to define and characterize CD4+ T cell responses from a broad repertoire of pathogenic arenaviruses (including lymphocytic choriomeningitis, Lassa, Guanarito, Junin, Machupo, Sabia, and Whitewater Arroyo viruses) and to provide determinants with the potential to be incorporated into a multivalent vaccine strategy. RESULTS: By inoculating HLA-DRB1*0101 transgenic mice with a panel of recombinant vaccinia viruses, each expressing a single arenavirus antigen, we identified 37 human HLA-DRB1*0101-restricted CD4+ T cell epitopes from the 7 antigenically distinct arenaviruses. We showed that the arenavirus-specific CD4+ T cell epitopes are capable of eliciting T cells with a propensity to provide help and protection through CD40L and polyfunctional cytokine expression. Importantly, we demonstrated that the set of identified CD4+ T cell epitopes provides broad, non-ethnically biased population coverage of all 7 arenavirus species targeted by our studies. CONCLUSIONS: The identification of CD4+ T cell epitopes, with promiscuous binding properties, derived from 7 different arenavirus species will aid in the development of a T cell-based vaccine strategy with the potential to target a broad range of ethnicities within the general population and to protect against both Old and New World arenavirus infection.

A multivalent and cross-protective vaccine strategy against arenaviruses associated with human disease.

Arenaviruses are the causative pathogens of severe hemorrhagic fever and aseptic meningitis in humans, for which no licensed vaccines are currently available. Pathogen heterogeneity within the Arenaviridae family poses a significant challenge for vaccine development. The main hypothesis we tested in the present study was whether it is possible to design a universal vaccine strategy capable of inducing simultaneous HLA-restricted CD8+ T cell responses against 7 pathogenic arenaviruses (including the lymphocytic choriomeningitis, Lassa, Guanarito, Junin, Machupo, Sabia, and Whitewater Arroyo viruses), either through the identification of widely conserved epitopes, or by the identification of a collection of epitopes derived from multiple arenavirus species. By inoculating HLA transgenic mice with a panel of recombinant vaccinia viruses (rVACVs) expressing the different arenavirus proteins, we identified 10 HLA-A02 and 10 HLA-A03-restricted epitopes that are naturally processed in human antigen-presenting cells. For some of these epitopes we were able to demonstrate cross-reactive CD8+ T cell responses, further increasing the coverage afforded by the epitope set against each different arenavirus species. Importantly, we showed that immunization of HLA transgenic mice with an epitope cocktail generated simultaneous CD8+ T cell responses against all 7 arenaviruses, and protected mice against challenge with rVACVs expressing either Old or New World arenavirus glycoproteins. In conclusion, the set of identified epitopes allows broad, non-ethnically biased coverage of all 7 viral species targeted by our studies.

ElliPro: a new structure-based tool for the prediction of antibody epitopes.

BACKGROUND: Reliable prediction of antibody, or B-cell, epitopes remains challenging yet highly desirable for the design of vaccines and immunodiagnostics. A correlation between antigenicity, solvent accessibility, and flexibility in proteins was demonstrated. Subsequently, Thornton and colleagues proposed a method for identifying continuous epitopes in the protein regions protruding from the protein's globular surface. The aim of this work was to implement that method as a web-tool and evaluate its performance on discontinuous epitopes known from the structures of antibody-protein complexes. RESULTS: Here we present ElliPro, a web-tool that implements Thornton's method and, together with a residue clustering algorithm, the MODELLER program and the Jmol viewer, allows the prediction and visualization of antibody epitopes in a given protein sequence or structure. ElliPro has been tested on a benchmark dataset of discontinuous epitopes inferred from 3D structures of antibody-protein complexes. In comparison with six other structure-based methods that can be used for epitope prediction, ElliPro performed the best and gave an AUC value of 0.732, when the most significant prediction was considered for each protein. Since the rank of the best prediction was at most in the top three for more than 70% of proteins and never exceeded five, ElliPro is considered a useful research tool for identifying antibody epitopes in protein antigens. ElliPro is available at http://tools.immuneepitope.org/tools/ElliPro. CONCLUSION: The results from ElliPro suggest that further research on antibody epitopes considering more features that discriminate epitopes from non-epitopes may further improve predictions. As ElliPro is based on the geometrical properties of protein structure and does not require training, it might be more generally applied for predicting different types of protein-protein interactions.

Immunomic analysis of the repertoire of T-cell specificities for influenza A virus in humans.

Continuing antigenic drift allows influenza viruses to escape antibody-mediated recognition, and as a consequence, the vaccine currently in use needs to be altered annually. Highly conserved epitopes recognized by effector T cells may represent an alternative approach for the generation of a more universal influenza virus vaccine. Relatively few highly conserved epitopes are currently known in humans, and relatively few epitopes have been identified from proteins other than hemagglutinin and nucleoprotein. This prompted us to perform a study aimed at identifying a set of human T-cell epitopes that would provide broad coverage against different virus strains and subtypes. To provide coverage across different ethnicities, seven different HLA supertypes were considered. More than 4,000 peptides were selected from a panel of 23 influenza A virus strains based on predicted high-affinity binding to HLA class I or class II and high conservancy levels. Peripheral blood mononuclear cells from 44 healthy human blood donors were tested for reactivity against HLA-matched peptides by using gamma interferon enzyme-linked immunospot assays. Interestingly, we found that PB1 was the major target for both CD4(+) and CD8(+) T-cell responses. The 54 nonredundant epitopes (38 class I and 16 class II) identified herein provided high coverage among different ethnicities, were conserved in the majority of the strains analyzed, and were consistently recognized in multiple individuals. These results enable further functional studies of T-cell responses during influenza virus infection and provide a potential base for the development of a universal influenza vaccine.

Immune epitope database analysis resource (IEDB-AR).

We present a new release of the immune epitope database analysis resource (IEDB-AR, http://tools.immuneepitope.org), a repository of web-based tools for the prediction and analysis of immune epitopes. New functionalities have been added to most of the previously implemented tools, and a total of eight new tools were added, including two B-cell epitope prediction tools, four T-cell epitope prediction tools and two analysis tools.

An analysis of the epitope knowledge related to Mycobacteria.

BACKGROUND: Tuberculosis, caused by the bacterium Mycobacterium tuberculosis, remains a leading cause of infectious disease morbidity and mortality, and is responsible for more than 2 million deaths a year. Reports about extremely drug resistant (XDR) strains have further heightened the sense of urgency for the development of novel strategies to prevent and treat TB. Detailed knowledge of the epitopes recognized by immune responses can aid in vaccine and diagnostics development, and provides important tools for basic research. The analysis of epitope data corresponding to M. tuberculosis can also identify gaps in our knowledge, and suggest potential areas for further research and discovery. The Immune Epitope Database (IEDB) is compiled mainly from literature sources, and describes a broad array of source organisms, including M. tuberculosis and other Mycobacterial species. DESCRIPTION: A comprehensive analysis of IEDB data regarding the genus Mycobacteria was performed. The distribution of antibody/B cell and T cell epitopes was analyzed in terms of their associated recognition cell type effector function and chemical properties. The various species, strains and proteins which the epitope were derived, were also examined. Additional variables considered were the host in which the epitopes were defined, the specific TB disease state associated with epitope recognition, and the HLA associated with disease susceptibility and endemic regions were also scrutinized. Finally, based on these results, standardized reference datasets of mycobacterial epitopes were generated. CONCLUSION: All current TB-related epitope data was cataloged for the first time from the published literature. The resulting inventory of more than a thousand different epitopes should prove a useful tool for the broad scientific community. Knowledge gaps specific to TB epitope data were also identified. In summary, few non-peptidic or post-translationally modified epitopes have been defined. Most importantly epitopes have apparently been defined from only 7% of all ORFs, and the top 30 most frequently studied protein antigens contain 65% of the epitopes, leaving the majority of M. tuberculosis genome unexplored. A lack of information related to the specific strains from which epitopes are derived is also evident. Finally, the generation of reference lists of mycobacterial epitopes should also facilitate future vaccine and diagnostic research.

Development of an epitope conservancy analysis tool to facilitate the design of epitope-based diagnostics and vaccines.

BACKGROUND: In an epitope-based vaccine setting, the use of conserved epitopes would be expected to provide broader protection across multiple strains, or even species, than epitopes derived from highly variable genome regions. Conversely, in a diagnostic and disease monitoring setting, epitopes that are specific to a given pathogen strain, for example, can be used to monitor responses to that particular infectious strain. In both cases, concrete information pertaining to the degree of conservancy of the epitope(s) considered is crucial. RESULTS: To assist in the selection of epitopes with the desired degree of conservation, we have developed a new tool to determine the variability of epitopes within a given set of protein sequences. The tool was implemented as a component of the Immune Epitope Database and Analysis Resources (IEDB), and is directly accessible at http://tools.immuneepitope.org/tools/conservancy. CONCLUSION: An epitope conservancy analysis tool was developed to analyze the variability or conservation of epitopes. The tool is user friendly, and is expected to aid in the design of epitope-based vaccines and diagnostics.

Chronic lymphocytic choriomeningitis virus infection actively down-regulates CD4+ T cell responses directed against a broad range of epitopes.

Activation of CD4(+) T cells helps establish and sustain CD8(+) T cell responses and is required for the effective clearance of acute infection. CD4-deficient mice are unable to control persistent infection and CD4(+) T cells are usually defective in chronic and persistent infections. We investigated the question of how persistent infection impacted pre-existing lymphocytic choriomeningitis virus (LCMV)-specific CD4(+) T cell responses. We identified class II-restricted epitopes from the entire set of open reading frames from LCMV Armstrong in BALB/c mice (H-2(d)) acutely infected with LCMV Armstrong. Of nine epitopes identified, six were restricted by I-A(d), one by I-E(d) and two were dually restricted by both I-A(d) and I-E(d) molecules. Additional experiments revealed that CD4(+) T cell responses specific for these epitopes were not generated following infection with the immunosuppressive clone 13 strain of LCMV. Most importantly, in peptide-immunized mice, established CD4(+) T cell responses to these LCMV CD4 epitopes as well as nonviral, OVA-specific responses were actively suppressed following infection with LCMV clone 13 and were undetectable within 12 days after infection, suggesting an active inhibition of established helper responses. To address this dysfunction, we performed transfer experiments using both the Smarta and OT-II systems. OT-II cells were not detected after clone 13 infection, indicating physical deletion, while Smarta cells proliferated but were unable to produce IFN-gamma, suggesting impairment of the production of this cytokine. Thus, multiple mechanisms may be involved in the impairment of helper responses in the setting of early persistent infection.

A quantitative analysis of the variables affecting the repertoire of T cell specificities recognized after vaccinia virus infection.

Many components contribute to immunodominance in the response to a complex virus, but their relative importance is unclear. This was addressed using vaccinia virus and HLA-A*0201 as the model system. A comprehensive analysis of 18 viral proteins recognized by CD8(+) T cell responses demonstrated that approximately one-fortieth of all possible 9- to 10-mer peptides were high-affinity HLA-A*0201 binders. Peptide immunization and T cell recognition data generated from 90 peptides indicated that about one-half of the binders were capable of eliciting T cell responses, and that one-seventh of immunogenic peptides are generated by natural processing. Based on these results, we estimate that vaccinia virus encodes approximately 150 dominant and subdominant epitopes restricted in by HLA-A*0201. However, of all these potential epitopes, only 15 are immunodominant and actually recognized in vivo during vaccinia virus infection of HLA-A*0201 transgenic mice. Neither peptide-binding affinity, nor complex stability, nor TCR avidity, nor amount of processed epitope appeared to strictly correlate with immunodominance status. Additional experiments suggested that vaccinia infection impairs the development of responses directed against subdominant epitopes. This suggested that additional factors, including immunoregulatory mechanisms, restrict the repertoire of T cell specificities after vaccinia infection by a factor of at least 10.