Convection-enhanced delivery of an anti-miR is well-tolerated, preserves anti-miR stability and causes efficient target de-repression: a proof of concept.

Over-expressed microRNAs (miRs) are promising new targets in glioblastoma (GBM) therapy. Inhibition of over-expressed miRs has been shown to diminish GBM proliferation, invasion and angiogenesis, indicating a significant therapeutic potential. However, the methods utilized for miR inhibition have had low translational potential. In clinical trials convection-enhanced delivery (CED) has been applied for local delivery of compounds in the brain. The aim of this study was to determine if safe and efficient miR inhibition was possible by CED of an anti-miR. We used a highly invasive GBM orthotopic xenograft model and targeted a well-validated miR, let-7a, with a 2'-O-methoxyethyl anti-miR with a combined phosphodiester/phosphorothioate backbone to establish an initial proof of concept. In vitro, anti-let-7a was delivered unassisted to the patient-derived T87 glioblastoma spheroid culture. In vivo, anti-let-7a or saline were administered by CED into orthotopic T87-derived tumors. After 1 month of infusion, tumors were removed and tumor mRNA levels of the target-gene High-mobility group AT-hook 2 (HMGA2) were determined. In vitro, 5 days inhibition was superior to 1 day at de-repressing the let-7a target HMGA2 and the inhibition was stable for 24 h. In vivo, anti-miR integrity was preserved in the pumps and no animals showed signs of severe adverse effects attributable to the anti-miR treatment. HMGA2 tumor level was significantly de-repressed in the anti-miR treated animals. The results showed-as an initial proof of concept-that miRs can be efficiently inhibited using CED delivery of anti-miR. The next step is to apply CED for anti-miR delivery focusing on key oncogenic miRs.

Shift of microRNA profile upon orthotopic xenografting of glioblastoma spheroid cultures.

Glioblastomas always recur despite surgery, radiotherapy and chemotherapy. A key player in the therapeutic resistance may be immature tumor cells with stem-like properties (TSCs) escaping conventional treatment. A group of promising molecular targets are microRNAs (miRs). miRs are small non-coding RNAs exerting post-transcriptional regulation of gene expression. In this study we aimed to identify over-expressed TSC-related miRs potentially amenable for therapeutic targeting. We used non-differentiated glioblastoma spheroid cultures (GSCs) containing TSCs and compared these to xenografts using a NanoString nCounter platform. This revealed 19 over-expressed miRs in the non-differentiated GSCs. Additionally, non-differentiated GSCs were compared to neural stem cells (NSCs) using a microarray platform. This revealed four significantly over-expressed miRs in the non-differentiated GSCs in comparison to the NSCs. The three most over-expressed miRs in the non-differentiated GSCs compared to xenografts were miR-126, -137 and -128. KEGG pathway analysis suggested the main biological function of these over-expressed miRs to be cell-cycle arrest and diminished proliferation. To functionally validate the profiling results suggesting association of these miRs with stem-like properties, experimental over-expression of miR-128 was performed. A consecutive limiting dilution assay confirmed a significantly elevated spheroid formation in the miR-128 over-expressing cells. This may provide potential therapeutic targets for anti-miRs to identify novel treatment options for GBM patients.

The Cancer Genome Atlas Analysis Predicts MicroRNA for Targeting Cancer Growth and Vascularization in Glioblastoma.

Using in silico analysis of The Cancer Genome Atlas (TCGA), we identified microRNAs associated with glioblastoma (GBM) survival, and predicted their functions in glioma growth and progression. Inhibition of two "risky" miRNAs, miR-148a and miR-31, in orthotopic xenograft GBM mouse models suppressed tumor growth and thereby prolonged animal survival. Intracranial tumors treated with uncomplexed miR-148a and miR-31 antagomirs exhibited reduced proliferation, stem cell depletion, and normalized tumor vasculature. Growth-promoting functions of these two miRNAs were, in part, mediated by the common target, the factor inhibiting hypoxia-inducible factor 1 (FIH1), and the downstream pathways involving hypoxia-inducible factor HIF1α and Notch signaling. Therefore, miR-31 and miR-148a regulate glioma growth by maintaining tumor stem cells and their niche, and providing the tumor a way to activate angiogenesis even in a normoxic environment. This is the first study that demonstrates intratumoral uptake and growth-inhibiting effects of uncomplexed antagomirs in orthotopic glioma.

Phase I randomized, observer-blinded, placebo-controlled study of a SARS-CoV-2 mRNA vaccine PTX-COVID19-B.

Access to vaccines against SARS-CoV-2 virus was limited in poor countries during the COVID-19 pandemic. Therefore, a low-cost mRNA vaccine, PTX-COVID19-B, was produced and evaluated in a Phase 1 trial. PTX-COVID19-B encodes Spike protein D614G variant without the proline-proline (986-987) mutation present in other COVID-19 vaccines. The aim of the study was to evaluate safety, tolerability, and immunogenicity of PTX-COVID19-B vaccine in healthy seronegative adults 18-64 years old. The trial design was observer-blinded, randomized, placebo-controlled, and tested ascending doses of 16-µg, 40-µg, or 100-µg in a total of 60 subjects who received two intramuscular doses, 4 weeks apart. Participants were monitored for solicited and unsolicited adverse events after vaccination and were provided with a Diary Card and thermometer to report any reactogenicity during the trial. Blood samples were collected on baseline, days 8, 28, 42, 90, and 180 for serum analysis of total IgG anti-receptor binding domain (RBD)/Spike titers by ELISA, and neutralizing antibody titers by pseudovirus assay. Titers in BAU/mL were reported as geometric mean and 95% CI per cohort. After vaccination, few solicited adverse events were observed and were mild to moderate and self-resolved within 48 h. The most common solicited local and systemic adverse event was pain at the injection site, and headache, respectively. Seroconversion was observed in all vaccinated participants, who showed high antibody titers against RBD, Spike, and neutralizing activity against the Wuhan strain. Neutralizing antibody titers were also detected against Alpha, Beta, and Delta variants of concerns in a dose dependent manner. All tested doses of PTX-COVID19-B were safe, well-tolerated, and provided a strong immunogenicity response. The 40-µg dose showed fewer adverse reactions than the 100-µg dose, and therefore was selected for a Phase 2 trial, which is currently ongoing.Clinical Trial Registration number: NCT04765436 (21/02/2021). ( https://clinicaltrials.gov/ct2/show/NCT04765436 ).

Onco-miR-21 Promotes Stat3-Dependent Gastric Cancer Progression.

MicroRNA-21 (miR-21) is a small, non-coding RNA overexpressed in gastric cancer and many other solid malignancies, where it exhibits both pro-and anti-tumourigenic properties. However, the pathways regulating miR-21 and the consequences of its inhibition in gastric cancer remain incompletely understood. By exploiting the spontaneous Stat3-dependent formation of inflammation-associated gastric tumors in Gp130F/F mice, we functionally established miR-21 as a Stat3-controlled driver of tumor growth and progression. We reconciled our discoveries by identifying several conserved Stat3 binding motifs upstream of the miR-21 gene promoter, and showed that the systemic administration of a miR-21-specific antisense oligonucleotide antagomir reduced the established gastric tumor burden in Gp130F/F mice. We molecularly delineated the therapeutic benefits of miR-21 inhibition with the functional restoration of PTEN in vitro and in vivo, alongside an attenuated epithelial-to-mesenchymal transition and the extracellular matrix remodeling phenotype of tumors. We corroborated our preclinical findings by correlating high STAT3 and miR-21 expression with the reduced survival probability of gastric cancer patients. Collectively, our results provide a molecular framework by which miR-21 mediates inflammation-associated gastric cancer progression, and establish miR-21 as a robust therapeutic target for solid malignancies characterized by excessive Stat3 activity.

Preclinical evaluation of a SARS-CoV-2 mRNA vaccine PTX-COVID19-B.

Safe and effective vaccines are needed to end the COVID-19 pandemic. Here, we report the preclinical development of a lipid nanoparticle­formulated SARS-CoV-2 mRNA vaccine, PTX-COVID19-B. PTX-COVID19-B was chosen among three candidates after the initial mouse vaccination results showed that it elicited the strongest neutralizing antibody response against SARS-CoV-2. Further tests in mice and hamsters indicated that PTX-COVID19-B induced robust humoral and cellular immune responses and completely protected the vaccinated animals from SARS-CoV-2 infection in the lung. Studies in hamsters also showed that PTX-COVID19-B protected the upper respiratory tract from SARS-CoV-2 infection. Mouse immune sera elicited by PTX-COVID19-B vaccination were able to neutralize SARS-CoV-2 variants of concern, including the Alpha, Beta, Gamma, and Delta lineages. No adverse effects were induced by PTX-COVID19-B in either mice or hamsters. Based on these results, PTX-COVID19-B was authorized by Health Canada to enter clinical trials in December 2020 with a phase 2 clinical trial ongoing.

STAT3 and STAT1 mediate IL-11-dependent and inflammation-associated gastric tumorigenesis in gp130 receptor mutant mice.

Deregulated activation of STAT3 is frequently associated with many human hematological and epithelial malignancies, including gastric cancer. While exaggerated STAT3 signaling facilitates an antiapoptotic, proangiogenic, and proproliferative environment for neoplastic cells, the molecular mechanisms leading to STAT3 hyperactivation remain poorly understood. Using the gp130(Y757F/Y757F) mouse model of gastric cancer, which carries a mutated gp130 cytokine receptor signaling subunit that cannot bind the negative regulator of cytokine signaling SOCS3 and is characterized by hyperactivation of the signaling molecules STAT1 and STAT3, we have provided genetic evidence that IL-11 promotes chronic gastric inflammation and associated tumorigenesis. Expression of IL-11 was increased in gastric tumors in gp130(Y757F/Y757F) mice, when compared with unaffected gastric tissue in wild-type mice, while gp130(Y757F/Y757F) mice lacking the IL-11 ligand-binding receptor subunit (IL-11Ralpha) showed normal gastric STAT3 activation and IL-11 expression and failed to develop gastric tumors. Furthermore, reducing STAT3 activity in gp130(Y757F/Y757F) mice, either genetically or by therapeutic administration of STAT3 antisense oligonucleotides, normalized gastric IL-11 expression and alleviated gastric tumor burden. Surprisingly, the genetic reduction of STAT1 expression also reduced gastric tumorigenesis in gp130(Y757F/Y757F) mice and coincided with reduced gastric inflammation and IL-11 expression. Collectively, our data have identified IL-11 as a crucial cytokine promoting chronic gastric inflammation and associated tumorigenesis mediated by excessive activation of STAT3 and STAT1.

Therapeutic suppression of translation initiation factor eIF4E expression reduces tumor growth without toxicity.

Expression of eukaryotic translation initiation factor 4E (eIF4E) is commonly elevated in human and experimental cancers, promoting angiogenesis and tumor growth. Elevated eIF4E levels selectively increase translation of growth factors important in malignancy (e.g., VEGF, cyclin D1) and is thereby an attractive anticancer therapeutic target. Yet to date, no eIF4E-specific therapy has been developed. Herein we report development of eIF4E-specific antisense oligonucleotides (ASOs) designed to have the necessary tissue stability and nuclease resistance required for systemic anticancer therapy. In mammalian cultured cells, these ASOs specifically targeted the eIF4E mRNA for destruction, repressing expression of eIF4E-regulated proteins (e.g., VEGF, cyclin D1, survivin, c-myc, Bcl-2), inducing apoptosis, and preventing endothelial cells from forming vessel-like structures. Most importantly, intravenous ASO administration selectively and significantly reduced eIF4E expression in human tumor xenografts, significantly suppressing tumor growth. Because these ASOs also target murine eIF4E, we assessed the impact of eIF4E reduction in normal tissues. Despite reducing eIF4E levels by 80% in mouse liver, eIF4E-specific ASO administration did not affect body weight, organ weight, or liver transaminase levels, thereby providing the first in vivo evidence that cancers may be more susceptible to eIF4E inhibition than normal tissues. These data have prompted eIF4E-specific ASO clinical trials for the treatment of human cancers.

Selective down-regulation of glioma-associated oncogene 2 inhibits the proliferation of hepatocellular carcinoma cells.

The sonic hedgehog (Shh) pathway contributes to the initiation and progression of tumors with various origins when aberrantly activated. In this study, we investigated if the Shh pathway is important for the proliferation of hepatocellular carcinoma (HCC) cells and also began to identify which components of the pathway play a pivotal role in the biology of HCC. Expression levels of components in the pathway were measured, and glioma-associated oncogene (Gli) 2 levels were found to be considerably higher in human HCC lines compared with normal liver. Gli2 levels were also higher in tumor tissue from HCC patients compared with normal liver. Antisense oligonucleotides (ASO) were used to specifically down-regulate Gli2, and this led to decreased proliferation of various HCC cell lines. However, inhibition of Gli1 and Gli3 with ASOs did not decrease proliferation in most HCC cell lines and inhibitors targeting the upstream components of the pathway, including smoothened (Smo), displayed antiproliferative effects in only a subset of HCC cell lines. Moreover, in cancer cells harboring Smo mutations or unresponsive to the Smo inhibitor 3-keto-N-aminoethylaminoethylcaproyldihydrocinnamoyl cyclopamine, the Gli2 ASO was still able to inhibit proliferation. The importance of Gli2 in HCC proliferation was further confirmed by the changes in expression levels of genes, such as Bcl-2, c-Myc, and p27, following suppression of Gli2 expression. Taken together, these results suggest that, among the Gli transcription factors, Gli2 plays a predominant role in the proliferation of HCC cells and the suppression of Gli2 expression may provide a useful therapeutic option for the treatment of HCC.

Competition for RISC binding predicts in vitro potency of siRNA.

Short interfering RNAs (siRNA) guide degradation of target RNA by the RNA-induced silencing complex (RISC). The use of siRNA in animals is limited partially due to the short half-life of siRNAs in tissues. Chemically modified siRNAs are necessary that maintain mRNA degradation activity, but are more stable to nucleases. In this study, we utilized alternating 2'-O-methyl and 2'-deoxy-2'-fluoro (OMe/F) chemically modified siRNA targeting PTEN and Eg5. OMe/F-modified siRNA consistently reduced mRNA and protein levels with equal or greater potency and efficacy than unmodified siRNA. We showed that modified siRNAs use the RISC mechanism and lead to cleavage of target mRNA at the same position as unmodified siRNA. We further demonstrated that siRNAs can compete with each other, where highly potent siRNAs can compete with less potent siRNAs, thus limiting the ability of siRNAs with lower potency to mediate mRNA degradation. In contrast, a siRNA with low potency cannot compete with a highly efficient siRNA. We established a correlation between siRNA potency and ability to compete with other siRNAs. Thus, siRNAs that are more potent inhibitors for mRNA destruction have the potential to out-compete less potent siRNAs indicating that the amount of a cellular component, perhaps RISC, limits siRNA activity.

High-content imaging analysis of the knockdown effects of validated siRNAs and antisense oligonucleotides.

High-content imaging (HCI) provides researchers with a powerful tool for understanding cellular processes. Although phenotypic analysis generated through HCI is a potent technique to determine the overall cellular effects of a given treatment, it frequently produces complex data sets requiring extensive interpretation. The authors developed statistical analyses to decrease the time spent to determine the outcome of each HCI assay and to better understand complex phenotypic changes. To test these tools, the authors performed a comparison experiment between 2 types of oligonucleotide-mediated gene silencing (OMGS), antisense oligonucleotides (ASOs), and short, double-stranded RNAs (siRNAs). Although similar in chemical structure, these 2 methods differ in cellular mechanism of action and off-target effects. Using a library of 50 validated ASOs and siRNAs to the same targets, the authors characterized the differential effects of these 2 technologies using a HeLa cell G2-M cell cycle assay. Although knockdown of a variety of targets by ASOs or siRNAs affected the cell cycle profile, few of those targets were affected by both ASOs and siRNAs. Distribution analysis of population changes induced through target knockdown led to the identification of targets that, when inhibited, could affect the G2-M transition in the cell cycle in a statistically significant manner. The distinctly different mechanisms of action of these 2 forms of gene silencing may help define the use of these treatments in both clinical and research environments.

Immune modulatory nanoparticle therapeutics for intracerebral glioma.

Background: Previously we showed therapeutic efficacy of unprotected miR-124 in preclinical murine models of glioblastoma, including in heterogeneous genetically engineered murine models by exploiting the immune system and thereby negating the need for direct tumor delivery. Although these data were promising, to implement clinical trials, we required a scalable formulation that afforded protection against circulatory RNases. Methods: We devised lipid nanoparticles that encapsulate and protect the miRs from degradation and provide enhanced delivery into the immune cell compartment and tested in vivo antitumor effects. Results: Treatment with nanoparticle-encapsulated miR-124, LUNAR-301, demonstrated a median survival exceeding 70 days, with an associated reversal of tumor-mediated immunosuppression and induction of immune memory. In both canine and murine models, the safety profile of LUNAR-301 was favorable. Conclusions: For the first time, we show that nanoparticles can direct a therapeutic response by targeting intracellular immune pathways. Although shown in the context of gliomas, this therapeutic approach would be applicable to other malignancies.

Identification of novel PPARgamma target genes in primary human adipocytes.

Adipogenesis is the process by which undifferentiated precursor cells differentiate into fat laden adipocytes. The nuclear proteins peroxisome proliferator-activated receptors (PPARs) play a central role in adipocyte differentiation. The goals of this study were to identify novel PPARgamma responsive genes and to determine their role in regulating human adipocyte differentiation. Affymetrix profiling of gene expression in human adipocytes identified about 1000 genes that were significantly up-regulated subsequent to induction of differentiation. PPARgamma expression was reduced prior to induction of differentiation using a novel, chemically modified antisense oligonucleotide. Affymetrix microarray profiling of these cells identified 278 statistically significantly down-regulated genes. Eight genes were found to contain previously documented PPARgamma recognition element (PPRE) in their upstream nucleotide (promoter) sequence. Four of these genes are novel and have not previously been characterized. Chromatin immuno-precipitation experiments confirmed the binding of PPARgamma to the PPRE of three of these genes. The ortholog of one of these genes, hypothetical protein FLJ 20920, has previously been reported to be involved in the control of body fat composition in Caenorhabditis elegans. Inhibition of expression of this protein was found to also inhibit differentiation of human adipocytes. MAST/MEME algorithm analysis was used to identify novel commonly occurring sequence motifs in the 5' upstream region of transcripts for subset of down-regulated genes, which were grouped according to their sequence similarities. A number of clusters were identified and the largest cluster contained similar motifs from 26 genes with the literature supporting 7 of the 26 genes as being involved in fatty acid metabolism or PPARgamma interaction.

Anti-miR delivery strategies to bypass the blood-brain barrier in glioblastoma therapy.

Small non-coding RNAs called miRNAs are key regulators in various biological processes, including tumor initiation, propagation, and metastasis in glioblastoma as well as other cancers. Recent studies have shown the potential for oncogenic miRNAs as therapeutic targets in glioblastoma. However, the application of antisense oligomers, or anti-miRs, to the brain is limited due to the blood-brain barrier (BBB), when administered in the traditional systemic manner. To induce a therapeutic effect in glioblastoma, anti-miR therapy requires a robust and effective delivery system to overcome this obstacle. To bypass the BBB, different delivery administration methods for anti-miRs were evaluated. Stereotaxic surgery was performed to administer anti-Let-7 through intratumoral (ITu), intrathecal (ITh), and intraventricular (ICV) routes, and each method's efficacy was determined by changes in the expression of anti-Let-7 target genes as well as by immunohistochemical analysis. ITu administration of anti-miRs led to a high rate of anti-miR delivery to tumors in the brain by both bolus and continuous administration. In addition, ICV administration, compared with ITu administration, showed a greater distribution of the miR across entire brain tissues. This study suggests that local administration methods are a promising strategy for anti-miR treatment and may overcome current limitations in the treatment of glioblastoma in preclinical animal models.

Therapeutic potential of targeting microRNA-10b in established intracranial glioblastoma: first steps toward the clinic.

MicroRNA-10b (miR-10b) is a unique oncogenic miRNA that is highly expressed in all GBM subtypes, while absent in normal neuroglial cells of the brain. miR-10b inhibition strongly impairs proliferation and survival of cultured glioma cells, including glioma-initiating stem-like cells (GSC). Although several miR-10b targets have been identified previously, the common mechanism conferring the miR-10b-sustained viability of GSC is unknown. Here, we demonstrate that in heterogeneous GSC, miR-10b regulates cell cycle and alternative splicing, often through the non-canonical targeting via 5'UTRs of its target genes, including MBNL1-3, SART3, and RSRC1. We have further assessed the inhibition of miR-10b in intracranial human GSC-derived xenograft and murine GL261 allograft models in athymic and immunocompetent mice. Three delivery routes for the miR-10b antisense oligonucleotide inhibitors (ASO), direct intratumoral injections, continuous osmotic delivery, and systemic intravenous injections, have been explored. In all cases, the treatment with miR-10b ASO led to targets' derepression, and attenuated growth and progression of established intracranial GBM. No significant systemic toxicity was observed upon ASO administration by local or systemic routes. Our results indicate that miR-10b is a promising candidate for the development of targeted therapies against all GBM subtypes.

Mcl-1 is required for Akata6 B-lymphoma cell survival and is converted to a cell death molecule by efficient caspase-mediated cleavage.

Enforced expression of the antiapoptotic Bcl-2 family protein Mcl-1 promotes lymphomagenesis in the mouse; however, the functional role of Mcl-1 in human B-cell lymphoma remains unclear. We demonstrate that Mcl-1 is widely expressed in malignant B-cells, and high-level expression of Mcl-1 is required for B-lymphoma cell survival, since transfection of Mcl-1-specific antisense oligodeoxynucleotides was sufficient to promote apoptosis in Akata6 lymphoma cells. Mcl-1 was efficiently cleaved by caspases at evolutionarily conserved aspartic acid residues in vitro, and during cisplatin-induced apoptosis in B-lymphoma cell lines and spontaneous apoptosis of primary malignant B-cells. Overexpression of the Mcl-1 cleavage product that accumulated during apoptosis was sufficient to kill cells. Therefore, Mcl-1 is an essential survival molecule for B-lymphoma cells and is cleaved by caspases to a death-promoting molecule during apoptosis. In contrast to Mcl-1, Bcl-2 and Bcl-XL were relatively resistant to caspase cleavage in vitro and in intact cells. Interfering with Mcl-1 function appears to be an effective means of inducing apoptosis in Mcl-1-positive B-cell lymphoma, and the unique sensitivity of Mcl-1 to caspase-mediated cleavage suggests an attractive strategy for converting it to a proapoptotic molecule.

Setting sights on the treatment of ocular angiogenesis using antisense oligonucleotides.

The application of antisense technology to study physiological and disease processes continues to mature. Antisense approaches are among the most direct means to use genomic sequence information. When developing therapeutics, applications range from early target validation in discovery to the therapeutic product. In this review, we describe the application of antisense oligonucleotides (ASOs) to identify genes that are important in controlling angiogenesis. High-throughput assays in vitro have been used to evaluate many gene targets. Genes that appear to be important in angiogenesis are then evaluated further in animal models of ocular angiogenesis. The ability of ASOs to reduce target-gene expression in the appropriate cells in the eye raises the possibility that this class of compounds could be used for target validation in vivo, and also be developed as a novel class of therapeutics in their own right.

MicroRNA-10b inhibition reduces E2F1-mediated transcription and miR-15/16 activity in glioblastoma.

MicroRNA-10b (miR-10b) is commonly elevated in glioblastoma (GBM), while not expressed in normal brain tissues. Targeted inhibition of miR-10b has pleiotropic effects on GBM derived cell lines, it reduces GBM growth in animal models, but does not affect normal neurons and astrocytes. This data raises the possibility of developing miR-10b-targeting GBM therapy. However, the mechanisms contributing to miR-10b-mediated glioma cell survival and proliferation are unexplored. We found that inhibition of miR-10b has distinct effects on specific glioma cell lines. In cells expressing high levels of tumor suppressor p21WAF1/Cip1, it represses E2F1-mediated transcription, leading to down-regulation of multiple E2F1 target genes encoding for S-phase specific proteins, epigenetic modulators, and miRNAs (e.g. miR-15/16), and thereby stalling progression through the S-phase of cell cycle. Subsequently, miR-15/16 activities are reduced and many of their direct targets are de-repressed, including ubiquitin ligase FBXW7 that destabilizes Cyclin E. Conversely, GBM cells expressing low p21 level, or after p21 knock-down, exhibit weaker or no E2F1 response to miR-10b inhibition. Comparative analysis of The Cancer Genome Atlas revealed a strong correlation between miR-10b and multiple E2F target genes in GBM and low-grade glioma. Taken together, these findings indicate that miR-10b regulates E2F1-mediated transcription in GBM, in a p21-dependent fashion.

Use of antisense oligonucleotides in functional genomics and target validation.

With the completion of sequencing of the human genome, a great deal of interest has been shifted toward functional genomics-based research for identification of novel drug targets for treatment of various diseases. The major challenge facing the pharmaceutical industry is to identify disease-causing genes and elucidate additional roles for genes of known functions. Gene functionalization and target validation are probably the most important steps involved in identifying novel potential drug targets. This review focuses on recent advances in antisense technology and its use for rapid identification and validation of new drug targets. The significance and applicability of this technology as a beginning of the drug discovery process are underscored by relevant cell culture-based assays and positive correlation in specific animal disease models. Some of the antisense inhibitors used to validate gene targets are themselves being developed as drugs. The current clinical trials based on such leads that were identified in a very short time further substantiate the importance of antisense technology-based functional genomics as an integral part of target validation and drug target identification.

Anti-miRs competitively inhibit microRNAs in Argonaute complexes.

MicroRNAs (miRNAs), small RNA molecules that post-transcriptionally regulate mRNA expression, are crucial in diverse developmental and physiological programs and their misregulation can lead to disease. Chemically modified oligonucleotides have been developed to modulate miRNA activity for therapeutic intervention in disease settings, but their mechanism of action has not been fully elucidated. Here we show that the miRNA inhibitors (anti-miRs) physically associate with Argonaute proteins in the context of the cognate target miRNA in vitro and in vivo. The association is mediated by the seed region of the miRNA and is sensitive to the placement of chemical modifications. Furthermore, the targeted miRNAs are stable and continue to be associated with Argonaute. Our results suggest that anti-miRs specifically associate with Argonaute-bound miRNAs, preventing association with target mRNAs, which leads to subsequent stabilization and thus increased expression of the targeted mRNAs.