Specific Antisense Oligonucleotides Research Articles

Circular RNA-encoded oncogenic PIAS1 variant blocks immunogenic ferroptosis by modulating the balance between SUMOylation and phosphorylation of STAT1

BackgroundThe clinical response rate to immune checkpoint blockade (ICB) therapy in melanoma remains low, despite its widespread use. Circular non-coding RNAs (circRNAs) are known to play a crucial role in cancer progression and may be a key factor limiting the effectiveness of ICB treatment.MethodsThe circRNAs that were downregulated after coadministration compared with single administration of PD-1 inhibitor administration were identified through RNA-seq and Ribo-seq, and thus the circPIAS1 (mmu_circ_0015773 in mouse, has_circ_0008378 in human) with high protein coding potential was revealed. Fluorescence in situ hybridization (FISH) assays were conducted to determine the localization of circPIAS1 in human and mouse melanoma cells, as well as its presence in tumor and adjacent tissues of patients. Validation through dual-luciferase reporter assay and LC–MS/MS confirmed the ability of circPIAS1 to encode a novel 108 amino acid polypeptide (circPIAS1-108aa). Specific antisense oligonucleotides (ASOs) targeting the junction site of circPIAS1 were developed to reduce its intracellular levels. Proliferation changes in melanoma cells were assessed using CCK8, EdU, and colony formation assays. The impact of circPIAS1-108aa on the ferroptosis process of melanoma cells was studied through GSH, MDA, and C11-BODIPY staining assays. Western Blot, Immunoprecipitation (IP), and Immunoprecipitation-Mass Spectrometry (IP-MS) techniques were employed to investigate the impact of circPIAS1-108aa on the P-STAT1/SLC7A11/GPX4 signaling pathway, as well as its influence on the balance between STAT1 SUMOylation and phosphorylation. Additionally, a melanoma subcutaneous transplanted tumor mouse model was utilized to examine the combined effect of reducing circPIAS1 levels alongside PD-1 inhibitor.ResultsCompared with the group treated with PD-1 inhibitor alone, circPIAS1 was significantly down-regulated in the coadministration group and demonstrated higher protein coding potential. CircPIAS1, primarily localized in the nucleus, was notably upregulated in tumor tissues compared to adjacent tissues, where it plays a crucial role in promoting cancer cell proliferation. This circRNA can encode a unique polypeptide consisting of 108 amino acids, through which it exerts its cancer-promoting function and impedes the effectiveness of ICB therapy. Mechanistically, circPIAS1-108aa hinders STAT1 phosphorylation by recruiting SUMO E3 ligase Ranbp2 to enhance STAT1 SUMOylation, thereby reactivating the transduction of the SLC7A11/GPX4 signaling pathway and restricting the immunogenic ferroptosis induced by IFNγ. Furthermore, the combination of ASO-circPIAS1 with PD-1 inhibitor effectively inhibits melanoma growth and significantly enhances the efficacy of immune drugs in vivo.ConclusionsOur study uncovers a novel mechanism regarding immune evasion in melanoma driven by a unique 108aa peptide encoded by circPIAS1 in melanoma that dramatically hinders immunogenic ferroptosis triggered by ICB therapy via modulating the balance between SUMOylation and phosphorylation of STAT1. This work reveals circPIAS1-108aa as a critical factor limiting the immunotherapeutic effects in melanoma and propose a promising strategy for improving ICB treatment outcomes.

Abstract B083: The splicing factor SMNDC1 facilitates alternative RNA splicing, contributing to therapy resistance in pancreatic cancer

Abstract Pancreatic ductal adenocarcinoma (PDAC) is among the most aggressive and lethal cancers, mainly due to its high resistance to existing treatments. The standard treatment for PDAC involves chemotherapy, either with Gemcitabine (GEM) combined with paclitaxel or the FOLFIRINOX regimen (5-fluorouracil (5FU), leucovorin, irinotecan, oxaliplatin). However, PDACs rapidly develop resistance to these therapies. Similar resistance is observed with new treatments targeting the KRAS-MAPK pathway, which is crucial in driving PDAC. Our preliminary studies have shown that Survival Motor Neuron Domain Containing 1 (SMNDC1) promotes PDAC tumor growth by facilitating exon retention, specifically cassette exon 4 (E4) of MAPK3, linking RNA splicing to KRAS-MAPK signaling. To uncover the mechanisms underlying PDAC therapeutic resistance, we have developed novel in vitro models of resistance to chemotherapy and MAPK inhibitors (MAPKi). Using these models, we found that SMNDC1 and MAPK3 E4 retention is significantly increased in GEM, 5FU (chemotherapy agents), Adagrasib, Sotorasib (KRAS inhibitors; KRASi G12C), and Selumetinib (MEK inhibitor; MEKi) resistant PDAC cells. Additionally, PDAC lines acutely treated (24h) with trametinib or selumetinib (MEKi) showed a doubling in the expression of SMNDC1 and MAPK3 E4 retention. Protein expression analysis via western blotting revealed that GEM, 5FU, Adagrasib, MRTX1133 (KRASi G12D), and Selumetinib resistant PDAC cells upregulated SMNDC1 expression compared to their sensitive counterparts. To determine the role of SMNDC1 in MAPK resistance, we tested SMNDC1 proficient and deficient PDAC cells with KRASi or MEKi. SMNDC1-deficient PDAC cells were more sensitive to these inhibitors than isogenic cells expressing SMNDC1. Furthermore, to show that increased E4 retention of MAPK3 drives resistance to KRASi or MEKi, we treated SMNDC1 proficient PDAC cells with these inhibitors and MAPK3 E4 specific antisense morpholino oligonucleotide (ASO), which increased sensitivity compared to cells treated with the inhibitor and control ASO. To determine if increased E4 retention is a resistance mechanism specific to therapies targeting upstream of ERK1, we treated PDAC cells, proficient and deficient in SMNDC1, with ERK inhibitors (MK8353, Temuterkib). No changes in sensitivity were observed, as these inhibitors target the ERK1 protein downstream of MAPK3 splicing. Additionally, in an orthogonal model, we found that mutant EGFR lung adenocarcinoma cells resistant to the third-generation tyrosine kinase EGFR inhibitor (Osimertinib) also had higher incorporation of E4 of MAPK3. This suggests that upstream inhibition of ERK in the MAPK pathway leads to increased MAPK3 E4 retention. These findings indicate that PDACs use a "splicing switch" to enhance ERK kinase activity, thereby resisting targeted therapies and chemotherapy in cancers driven by mutant KRAS-MAPK signaling pathways. Citation Format: Md Afjalus Siraj, Yushan Zhang, Deanne Yugawa, Gilbert Giri, Prabir Chakraborty, Grant Goda, Geeta Rao, Daniel Dominguez, Stefan Kubicek, Resham Bhattacharya, Luisa Escobar-Hoyos, Priyabrata Mukherjee. The splicing factor SMNDC1 facilitates alternative RNA splicing, contributing to therapy resistance in pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr B083.

Bromodomain-containing 4 is a positive regulator of the inflammatory cytokine response in the gut.

Bromodomain-containing protein 4 (BRD4), one of the components of the bromodomain and extraterminal domain (BET) family, is a transcriptional and epigenetic regulator of cellular proliferation and cytokine production. In this study, we assessed whether BRD4 regulates the cytokine response in inflammatory bowel diseases (IBD). BRD4 expression was analyzed in intestinal mucosal samples of patients with ulcerative colitis (UC), patients with Crohn's disease (CD), normal controls (CTRs), and mice with chemically-induced colitis by real-time PCR, Western blotting, and confocal microscopy. Cytokine production was evaluated in lamina propria mononuclear cells (LPMCs) of IBD patients and mucosal tissues of colitic mice treated with BRD4 inhibitors. Finally, we evaluated the effect of JQ1, an inhibitor of the BRD4 signaling pathway, on the course of murine colitis. BRD4 RNA and protein expression was up-regulated in the inflamed mucosa of patients with UC and patients with CD as compared to the uninvolved areas of the same patients and CTRs, and in the inflamed colon of colitic mice. Knockdown of BRD4 with a specific antisense oligonucleotide in IBD LPMCs led to reduced expression of TNF-α, IL-6, IFN-γ, and IL-17A. Administration of JQ1 to colitic mice inhibited the inflammatory cytokine response and attenuated the ongoing colitis. This is the first study showing the up-regulation of BRD4 in IBD and suggesting the role of such a protein in the positive control of the inflammatory cytokine response in the gut.

SMAD7 Sustains XIAP Expression and Migration of Colorectal Carcinoma Cells.

The reorganization of the cell cytoskeleton and changes in the content of cell adhesion molecules are crucial during the metastatic spread of tumor cells. Colorectal cancer (CRC) cells express high SMAD7, a protein involved in the control of CRC cell growth. In the present study, we evaluated whether SMAD7 regulates the cytoskeleton reorganization and dynamics in CRC. Knockdown of SMAD7 with a specific antisense oligonucleotide (AS) in HCT116 and DLD1, two human CRC cell lines, reduced the migration rate and the content of F-ACTIN filaments. A gene array, real-time PCR, and Western blotting of SMAD7 AS-treated cells showed a marked down-regulation of the X-linked inhibitor of apoptosis protein (XIAP), a member of the inhibitor of apoptosis family, which has been implicated in cancer cell migration. IL-6 and IL-22, two cytokines that activate STAT3, enhanced XIAP in cancer cells, and such induction was attenuated in SMAD7-deficient cells. Finally, in human CRC, SMAD7 mRNA correlated with XIAP expression. Our data show that SMAD7 positively regulates XIAP expression and migration of CRC cells, and suggest a mechanism by which SMAD7 controls the architecture components of the CRC cell cytoskeleton.

Antisense oligonucleotide conjugated gold nanoconstructs-based electrochemical biosensor for detection of SARS-CoV-2

Nanoconstructs of gold nanoparticles (AuNPs) conjugated with SARS-CoV-2 specific antisense oligonucleotides (ASO) have been utilized to develop sensitive electrochemical nucleic acid biosensor for the detection of SARS-CoV-2 RNA. AuNPs were prepared through a one-pot synthesis method by utilizing Poly-L-Lysine (PLL) biopolymer and as synthesised AuNP were characterized by various analytical techniques such as UV–Vis spectroscopy, X-ray Diffraction (XRD) analysis, Fourier Transform Infra-Red spectroscopy (FT-IR), zeta potential, and Transmission Electron Microscopy (TEM). Poly-L-Lysine functionalized AuNPs (PLL-AuNPs) nanoconstructs platform was employed for immobilization of SARS-CoV-2 specific antisense oligonucleotides (ASO-conjugated PLL-AuNPs) via electrostatic interactions. The PLL-AuNPs were drop casted on glassy carbon electrode (GCE) following immobilization of ASO for fabrication of electrochemical biosensor. The ASO-conjugated PLL-AuNPs nanoconstructs were characterized by cyclic voltammetry (CV), differential pulse voltammetry (DPV), and electrochemical impedance spectroscopy (EIS) techniques. The responsiveness of ASO-conjugated PLL-AuNPs nanoconstructs in presence SARS-CoV-2 RNA was monitored using the DPV, SWV and EIS technique, where methylene blue was employed as an electrochemical indicator for DNA-RNA hybridization detection. The biosensor exhibits a detection range for SARS-CoV-2 RNA infection ranging from 0 to 100 nM, with a limit of detection at 30.2 nM. The electrode, modified with ASO-conjugated PLL-AuNPs, was employed for the detection of SARS-CoV-2 RNA from clinical samples collected from COVID-19-positive individuals.

Human angiotensin-converting enzyme 2–specific antisense oligonucleotides reduce infection with SARS-CoV-2 variants

BackgroundThe Spike protein mutation of SARS-CoV-2 led to decreased protective effect of various vaccines and monoclonal antibodies, suggesting that blocking SARS-CoV-2 infection by targeting host factors would make the therapy more resilient against virus mutations. Angiotensin converting enzyme 2 (ACE2) is the host receptor of SARS-CoV-2 and its variants, as well as many other coronaviruses. Down-regulation of ACE2 expression in the respiratory tract may prevent viral infection. Antisense oligonucleotides (ASOs) can be rationally designed based on sequence data, require no delivery system, and can be administered locally. ObjectiveWe sought to design ASOs that can block SARS-CoV-2 by down-regulating ACE2 in human airway. MethodsACE2-targeting ASOs were designed using a bioinformatic method and screened in cell lines. Human primary nasal epithelial cells cultured at the air-liquid interface and humanized ACE2 mice were used to detect the ACE2 reduction levels and the safety of ASOs. ASOs pretreated nasal epithelial cells and mice were infected and then used to detect the viral infection levels. ResultsASOs reduced ACE2 expression on mRNA and protein level in cell lines and in human nasal epithelial cells. Furthermore they efficiently suppressed virus replication of three different SARS-CoV-2 variants in human nasal epithelial cells. In vivo, ASOs also down-regulated human ACE2 in humanized ACE2 mice and thereby reduced viral load, histopathological changes in lungs, and they increased survival of mice. ConclusionACE2-targeting ASOs can effectively block SARS-COV-2 infection. Our study provides a new approach for blocking SARS-CoV-2 and other ACE2-targeting virus in high-risk populations.

A robust bioplatform based on DNA-gated nanoscale porous carbon derived from a metal-organic framework for specific detection of Herpes simplex virus type 1

In this study, a highly efficient sensing platform based on metal-organic framework-derived porous carbon (MOF-PC) and bio-gatekeeper strategy was designed for the sensitive detection of Herpes simplex virus type 1 (HSV-1). To realize the aim above, MOF-PC was obtained through the carbonization of the zeolitic imidazolate framework (ZIF-8). The electroactive dye methylene blue (MB) was then captured as a signal probe in the MOF-PC pores. Specifically, HSV-1 highly specific antisense ssDNA oligonucleotide was connected to MOF-PC through π-stacking interaction as a gatekeeper and sealed up the pores of MOF-PC. By adding the targets (synthetic ssDNA target or total genome HSV-1 DNA) and hybridization between the immobilized specific antisense ssDNA oligonucleotides and the targets, the double-stranded DNA (dsDNA) was separated from the MOF-PC surface due to the low affinity of the MOF-PC to the dsDNA. Meanwhile, MBs were released from the opened holes of MOF-PC. Therefore, the intensity of MB peak current in the absence of a target was high and then decreased by releasing and washing of MB molecules. The MB@MOF-PC responded linearly to synthetic ssDNA target in the range from 10−18 to 10−8 M and isolated total genome HSV-1 DNA in the range from 102 to 108 copies/mL. The detection limits of 0.79 aM and 83.4 copies/mL were obtained for the synthetic ssDNA target and total genome HSV-1 DNA, respectively.

Inhibition of MER proto-oncogene tyrosine kinase by an antisense oligonucleotide enhances treatment efficacy of immunoradiotherapy

BackgroundThe combination of radiotherapy and immunotherapy (immunoradiotherapy) has been increasingly used for treating a wide range of cancers. However, some tumors are resistant to immunoradiotherapy. We have previously shown that MER proto-oncogene tyrosine kinase (MerTK) expressed on macrophages mediates resistance to immunoradiotherapy. We therefore sought to develop therapeutics that can mitigate the negative impact of MerTK. We designed and developed a MerTK specific antisense oligonucleotide (ASO) and characterized its effects on eliciting an anti-tumor immune response in mice.Methods344SQR cells were injected into the right legs on day 0 and the left legs on day 4 of 8-12 weeks old female 129sv/ev mice to establish primary and secondary tumors, respectively. Radiation at a dose of 12 Gy was given to the primary tumors on days 8, 9, and 10. Mice received either anti-PD-1, anti-CTLA-4 or/and MerTK ASO starting from day 1 post tumor implantation. The composition of the tumor microenvironment and the level of MerTK on macrophages in the tumor were evaluted by flow cytometry. The expression of immune-related genes was investigated with NanoString. Lastly, the impact of MerTK ASO on the structure of the eye was histologically evaluated.ResultsRemarkably, the addition of MerTK ASO to XRT+anti-PD1 and XRT+anti-CTLA4 profoundly slowed the growth of both primary and secondary tumors and significantly extended survival. The ASO significantly reduced the expression of MerTK in tumor-associated macrophages (TAMs), reprograming their phenotype from M2 to M1. In addition, MerTK ASO increased the percentage of Granzyme B+ CD8+ T cells in the secondary tumors when combined with XRT+anti-CTLA4. NanoString results demonstrated that the MerTK ASO favorably modulated immune-related genes for promoting antitumor immune response in secondary tumors. Importantly, histological analysis of eye tissues demonstrated that unlike small molecules, the MerTK ASO did not produce any detectable pathology in the eyes.ConclusionsThe MerTK ASO can significantly downregulate the expression of MerTK on TAMs, thereby promoting antitumor immune response. The combination of MerTK ASO with immunoradiotherapy can safely and significantly slow tumor growth and improve survival.Graphical

Biomimetic nanodecoys deliver cholesterol-modified heteroduplex oligonucleotide to target dopaminergic neurons for the treatment of Parkinson's disease

Parkinson's disease (PD) is the second most common neurodegenerative disorder characterized by the accumulation of α-synuclein (α-syn) aggregates called Lewy bodies leading to the gradual loss of dopaminergic (DA) neurons in the substantia nigra. Although α-syn expression can be attenuated by antisense oligonucleotides (ASOs) and heteroduplex oligonucleotide (HDO) by intracerebroventricular (ICV) injection, the challenge to peripheral targeted delivery of oligonucleotide safely and effectively into DA neurons remains unresolved. Here, we designed a new DNA/DNA double-stranded (complementary DNA, coDNA) molecule with cholesterol conjugation (Chol-HDO (coDNA)) based on an α-syn-ASO sequence and evaluated its silence efficiency. Further, Chol-HDO@LMNPs, Chol-HDO-loaded, cerebrovascular endothelial cell membrane with DSPE-PEG2000-levodopa modification (L-DOPA-CECm)-coated nanoparticles (NPs), were developed for the targeted treatment of PD by tail intravenous injection. CECm facilitated the blood-brain barrier (BBB) penetration of NPs, together with cholesterol escaped from reticuloendothelial system uptake, as well as L-DOPA was decarboxylated into dopamine which promoted the NPs toward the PD site for DA neuron regeneration. The behavioral tests demonstrated that the nanodecoys improved the efficacy of HDO on PD mice. These findings provide insights into the development of biomimetic nanodecoys loading HDO for precise therapy of PD. Statement of significanceThe accumulation of α-synuclein (α-syn) aggregates is a hallmark of PD. Our previous study designed a specific antisense oligonucleotide (ASO) targeting human SNCA, but the traumatic intracerebroventricular (ICV) is not conducive to clinical application. Here, we further optimize the ASO by creating a DNA/DNA double-stranded molecule with cholesterol-conjugated, named Chol-HDO (coDNA), and develop a DA-targeted biomimetic nanodecoy Chol-HDO@LMNPs by engineering cerebrovascular endothelial cells membranes (CECm) with DSPE-PEG2000 and L-DOPA. The in vivo results demonstrated that tail vein injection of Chol-HDO@LMNPs could target DA neurons in the brain and ameliorate motor deficits in a PD mouse model. This investigation provides a promising peripheral delivery platform of L-DOPA-CECm nanodecoy loaded with a new Chol-HDO (coDNA) targeting DA neurons in PD therapy.

Cellular and subcellular localization of Rab10 and phospho-T73 Rab10 in the mouse and human brain

Autosomal dominant pathogenic mutations in Leucine-rich repeat kinase 2 (LRRK2) cause Parkinson’s disease (PD). The most common mutation, G2019S-LRRK2, increases the kinase activity of LRRK2 causing hyper-phosphorylation of its substrates. One of these substrates, Rab10, is phosphorylated at a conserved Thr73 residue (pRab10), and is one of the most abundant LRRK2 Rab GTPases expressed in various tissues. The involvement of Rab10 in neurodegenerative disease, including both PD and Alzheimer’s disease makes pinpointing the cellular and subcellular localization of Rab10 and pRab10 in the brain an important step in understanding its functional role, and how post-translational modifications could impact function. To establish the specificity of antibodies to the phosphorylated form of Rab10 (pRab10), Rab10 specific antisense oligonucleotides were intraventricularly injected into the brains of mice. Further, Rab10 knock out induced neurons, differentiated from human induced pluripotent stem cells were used to test the pRab10 antibody specificity. To amplify the weak immunofluorescence signal of pRab10, tyramide signal amplification was utilized. Rab10 and pRab10 were expressed in the cortex, striatum and the substantia nigra pars compacta. Immunofluorescence for pRab10 was increased in G2019S-LRRK2 knockin mice. Neurons, astrocytes, microglia and oligodendrocytes all showed Rab10 and pRab10 expression. While Rab10 colocalized with endoplasmic reticulum, lysosome and trans-Golgi network markers, pRab10 did not localize to these organelles. However, pRab10, did overlap with markers of the presynaptic terminal in both mouse and human cortex, including α-synuclein. Results from this study suggest Rab10 and pRab10 are expressed in all brain areas and cell types tested in this study, but pRab10 is enriched at the presynaptic terminal. As Rab10 is a LRRK2 kinase substrate, increased kinase activity of G2019S-LRRK2 in PD may affect Rab10 mediated membrane trafficking at the presynaptic terminal in neurons in disease.

Targeting Cx43 to Reduce the Severity of Pressure Ulcer Progression.

In the skin, repeated incidents of ischemia followed by reperfusion can result in the breakdown of the skin and the formation of a pressure ulcer. Here we gently applied paired magnets to the backs of mice to cause ischemia for 1.5 h and then removed them to allow reperfusion. The sterile inflammatory response generated within 4 h causes a stage 1 pressure ulcer with an elevation of the gap junction protein Cx43 in the epidermis. If this process is repeated the insult will result in a more severe stage 2 pressure ulcer with a breakdown of the epidermis 2-3 days later. After a single pinch, the elevation of Cx43 in the epidermis is associated with the inflammatory response with an increased number of neutrophils, HMGB1 (marker of necrosis) and RIP3 (responsible for necroptosis). Delivering Cx43 specific antisense oligonucleotides sub-dermally after a single insult, was able to significantly reduce the elevation of epidermal Cx43 protein expression and reduce the number of neutrophils and prevent the elevation of HMGB1 and RIP3. In a double pinch model, the Cx43 antisense treatment was able to reduce the level of inflammation, necroptosis, and the extent of tissue damage and progression to an open wound. This approach may be useful in reducing the progression of stage 1 pressure ulcers to stage 2.

Specific intracellular retention of circSKA3 promotes colorectal cancer metastasis by attenuating ubiquitination and degradation of SLUG

Our previous study demonstrated that tumor-suppressor circular RNAs (circRNAs) can be specifically secreted outside of colorectal cancer (CRC) cells within exosomes to maintain tumor cell fitness. However, whether tumor-driving circRNAs can be specifically retained in cells to facilitate tumor progression remains unknown. In this study, circRNA-seq showed that circSKA3 was significantly upregulated in CRC tissues but downregulated in serum samples from CRC patients. In addition, circSKA3 promoted CRC progression in vitro and in vivo and was retained in CRC cells via a specific cellmotif element. Interestingly, the cellmotif element was also the site of interaction of circSKA3 with SLUG, which inhibited SLUG ubiquitination degradation and promoted CRC epithelial–mesenchymal transition (EMT). Moreover, FUS was identified as a key circularization regulator of circSKA3 that bound to the key element. Finally, we designed and synthesized specific antisense oligonucleotides (ASOs) targeting circularization and cellmotif elements, which repressed circSKA3 expression, abolished the SLUG–circSKA3 interaction, and further inhibited CRC EMT and metastasis in vitro and in vivo.

Targeted suppression of mTORC2 reduces seizures across models of epilepsy

Epilepsy is a neurological disorder that poses a major threat to public health. Hyperactivation of mTOR complex 1 (mTORC1) is believed to lead to abnormal network rhythmicity associated with epilepsy, and its inhibition is proposed to provide some therapeutic benefit. However, mTOR complex 2 (mTORC2) is also activated in the epileptic brain, and little is known about its role in seizures. Here we discover that genetic deletion of mTORC2 from forebrain neurons is protective against kainic acid-induced behavioral and EEG seizures. Furthermore, inhibition of mTORC2 with a specific antisense oligonucleotide robustly suppresses seizures in several pharmacological and genetic mouse models of epilepsy. Finally, we identify a target of mTORC2, Nav1.2, which has been implicated in epilepsy and neuronal excitability. Our findings, which are generalizable to several models of human seizures, raise the possibility that inhibition of mTORC2 may serve as a broader therapeutic strategy against epilepsy.

Reduction of Tumor Growth with RNA-Targeting Treatment of the NAB2-STAT6 Fusion Transcript in Solitary Fibrous Tumor Models.

Solitary fibrous tumor (SFT) is a rare soft-tissue sarcoma. This nonhereditary cancer is the result of an environmental intrachromosomal gene fusion between NAB2 and STAT6 on chromosome 12, which fuses the activation domain of STAT6 with the repression domain of NAB2. Currently there is not an approved chemotherapy regimen for SFTs. The best response on available pharmaceuticals is a partial response or stable disease for several months. The purpose of this study is to investigate the potential of RNA-based therapies for the treatment of SFTs. Specifically, in vitro SFT cell models were engineered to harbor the characteristic NAB2-STAT6 fusion using the CRISPR/SpCas9 system. Cell migration as well as multiple cancer-related signaling pathways were increased in the engineered cells as compared to the fusion-absent parent cells. The SFT cell models were then used for evaluating the targeting efficacies of NAB2-STAT6 fusion-specific antisense oligonucleotides (ASOs) and CRISPR/CasRx systems. Our results showed that fusion specific ASO treatments caused a 58% reduction in expression of fusion transcripts and a 22% reduction in cell proliferation after 72 h in vitro. Similarly, the AAV2-mediated CRISPR/CasRx system led to a 59% reduction in fusion transcript expressions in vitro, and a 55% reduction in xenograft growth after 29 days ex vivo.

A wave of deep intronic mutations in X-linked Alport syndrome

X-linked Alport syndrome (XLAS) is an inherited kidney disease caused exclusively by pathogenic variants in the COL4A5 gene. In 10-20% of cases, DNA sequencing of COL4A5 exons or flanking regions cannot identify molecular causes. Here, our objective was to use a transcriptomic approach to identify causative events in a group of 19 patients with XLAS without identified mutation by Alport gene panel sequencing. Bulk RNAseq and/or targeted RNAseq using a capture panel of kidney genes was performed. Alternative splicing events were compared to those of 15 controls by a developed bioinformatic score. When using targeted RNAseq, COL4A5 coverage was found to be 23-fold higher than with bulk RNASeq and revealed 30 significant alternative splicing events in 17 of the 19 patients. After computational scoring, a pathogenic transcript was found in all patients. A causative variant affecting COL4A5 splicing and absent in the general population was identified in all cases. Altogether, we developed a simple and robust method for identification of aberrant transcripts due to pathogenic deep-intronic COL4A5 variants. Thus, these variants, potentially targetable by specific antisense oligonucleotide therapies, were found in a high percentage of patients with XLAS in whom pathogenic variants were missed by conventional DNA sequencing.

An essential role of adenosine deaminase acting on RNA 1 in coeliac disease mucosa.

Type I interferons (IFNs) are highly expressed in the gut mucosa of celiac disease (CD) gut mucosa and stimulates immune response prompted by gluten ingestion, but the processes that maintain the production of these inflammatory molecules are not well understood. Adenosine deaminase acting on RNA 1 (ADAR1), an RNA-editing enzyme, plays a crucial role in inhibiting self or viral RNAs from activating auto-immune mediated responses, most notably within the type-I IFN production pathway. The aim of this study was to assess whether ADAR1 could contribute to the induction and/or progression of gut inflammation in patients with celiac disease. ADAR1 expression was assessed by Real time PCR and Western blotting in duodenal biopsy taken from inactive and active celiac disease (CD) patients and normal controls (CTR). To analyze the role of ADAR1 in inflamed CD mucosa, lamina propria mononuclear cells (LPMC) were isolated from inactive CD and ADAR1 was silenced in with a specific antisense oligonucleotide (AS) and then incubated with a synthetic analogue of viral dsRNA (poly I:C). IFN-inducing pathways (IRF3, IRF7) in these cells were evaluated with Western blotting and inflammatory cytokines were evaluated with flow cytometry. Lastly, the role of ADAR1 was investigated in a mouse model of poly I:C-driven small intestine atrophy. Reduced ADAR1 expression was seen in duodenal biopsies compared to inactive CD and normal controls. Ex vivo organ cultures of duodenal mucosal biopsies, taken from inactive CD patients, stimulated with a peptic-tryptic digest of gliadin displayed a decreased expression of ADAR1. ADAR1 silencing in LPMC stimulated with a synthetic analogue of viral dsRNA strongly boosted the activation of IRF3 and IRF7 and the production of type-I IFN, TNF-α and IFN-γ. Administration of ADAR1 antisense but not sense oligonucleotide to mice with poly I:C-induced intestinal atrophy, significantly increased gut damage and inflammatory cytokines production. These data show that ADAR1 is an important regulator of intestinal immune homeostasis and demonstrate that defective ADAR1 expression could provide to amplifying pathogenic responses in CD intestinal mucosa.

Nuclear factor 1 X-type-associated regulation of myogenesis in developing mouse tongue

ObjectivesThe tongue contains skeletal myofibers that differ from those in the trunk, limbs, and other orofacial muscles. However, the molecular basis of myogenic differentiation in the tongue muscles remains unclear. In this study, we conducted comprehensive gene expression profiling of the developing murine tongue. MethodsTongue primordia were dissected from mouse embryos at embryonic day (E)10.5–E18.5, while myogenic markers were detected via microarray analysis and quantitative polymerase chain reaction (PCR). In addition to common myogenic regulatory factors such as Myf5, MyoD, myogenin, and Mrf4, we focused on Nfix, which acts as a unique molecular switch triggering the shift from embryonic to fetal myoblast lineage during limb myogenesis. Nfix inhibition was performed using a specific antisense oligonucleotide in the organ culture of tongue primordia. ResultsMicroarray and ingenuity pathway analyses confirmed the significant upregulation of myogenic signaling molecules, including Nfix, associated with the differentiation of myoblasts from myogenic progenitor cells during E10.5–E11.5. Quantitative PCR confirmed that Nfix expression started at E10.5 and peaked at E14.5. Fetal myoblast-specific genes, such as Mck and Myh8, were upregulated after E14.5, whereas embryonic myoblast-specific genes, such as Myh3 and Myh7, were downregulated. When Nfix was inhibited in the organ culture of tongue primordia, subtle morphological differences were noted in the tongue. Such an observation was only noted in the cultures of E10.5-derived tongue primordia. ConclusionsThese results reveal the contribution of Nfix to tongue myogenesis. Nfix expression during early tongue development may play a vital role in tongue muscle development.

Elevation of SHANK3 Levels by Antisense Oligonucleotides Directed Against the 3'-UTR of the Human SHANK3 mRNA.

SHANK3 is a member of the SHANK family of scaffolding proteins that localize to the postsynaptic density of excitatory synapses. Mutations within the SHANK3 gene or SHANK3 haploinsufficiency is thought to be one of the major causes for Phelan-McDermid Syndrome (PMDS) that is characterized by a broad spectrum of autism-related behavioral alterations. Several approaches have already been proposed to elevate SHANK3 protein levels in PMDS patients like transcriptional activation or inhibition of SHANK3 degradation. We undertook a systematic screening approach and tested whether defined antisense oligonucleotides (ASOs) directed against the 3' untranslated region (3'-UTR) of the human SHANK3 mRNA are suitable to elevate SHANK3 protein levels. Using human induced pluripotent stem cells (hiPSCs) and hiPSCs-derived motoneurons from controls and PMDS patients we eventually identified two 18 nucleotide ASOs (ASO 4-5.2-4 and 4-5.2-6) that were able to increase SHANK3 protein levels in vitro by about 1.3- to 1.6-fold. These findings were confirmed by co-transfection of the identified ASOs with a GFP-SHANK3-3'-UTR construct in HEK293T cells using GFP protein expression as read-out. Based on these results we propose a novel approach to elevate SHANK3 protein concentrations by 3'-UTR specific ASOs. Further research is needed to test the suitability of SHANK3-specific ASOs as pharmacological compounds also in vivo.

The Influence of Plasma Prekallikrein Oligonucleotide Antisense Therapy on Coagulation and Fibrinolysis Assays: A Post-hoc Analysis.

Background Congenital prekallikrein deficiency has been associated with increased risk of thrombosis in a few uncontrolled studies. Prekallikrein levels were reduced by 36-94% (median 75%) in a phase 2 study with the prekallikrein specific antisense oligonucleotide, donidalorsen, in patients with hereditary angioedema (HAE). Objectives To estimate the effects of plasma prekallikrein reduction on coagulation and fibrinolysis. Methods Plasma samples were obtained from 16 HAE patients treated with donidalorsen and six placebo-treated patients. Calibrated automated thrombogram (CAT), clot lysis time, high-molecular-weight kininogen activity, factor XI activity, prothrombin fragment 1+2 (F1+2), thrombin-antithrombin complexes, D-dimer, plasminogen activity, plasmin-α2-antiplasmin complexes, and α2-antiplasmin activity were measured before and after four months of treatment. Results No significant changes following donidalorsen treatment were observed between baseline and after four months of treatment: CAT lag time 3.0 min and 3.0 min, CAT peak thrombin concentration 109% and 129%, CAT endogenous thrombin potential 98% and 108%, factor XI activity 110% and 115%, F1+2 levels 251 pMol/L and 161 pMol/L, thrombin-antithrombin complexes 2.6 µg/L and 1.9 µg/L, high-molecular-weight kininogen activity 87% and 93%, clot lysis time 92% and 99%, D-dimer levels 0.65 µg/L and 0.36 µg/L, plasminogen activity 116% and 125%, plasmin-α2-antiplasmin complexes 533 ng/mL and 328 ng/mL, and α2-antiplasmin activity 122% and 127%, respectively. There were also no differences between donidalorsen and placebo treatment. Conclusions Reduction of plasma prekallikrein by donidalorsen in HAE patients neither affected thrombin formation nor fibrinolytic activity. Our data suggest that partial plasma prekallikrein reduction does not influence thrombotic risk.

LINE-1 RNA causes heterochromatin erosion and is a target for amelioration of senescent phenotypes in progeroid syndromes.

Constitutive heterochromatin is responsible for genome repression of DNA enriched in repetitive sequences, telomeres, and centromeres. During physiological and pathological premature aging, heterochromatin homeostasis is profoundly compromised. Here, we showed that LINE-1 (Long Interspersed Nuclear Element-1; L1) RNA accumulation was an early event in both typical and atypical human progeroid syndromes. L1 RNA negatively regulated the enzymatic activity of the histone-lysine N-methyltransferase SUV39H1 (suppression of variegation 3-9 homolog 1), resulting in heterochromatin loss and onset of senescent phenotypes in vitro. Depletion of L1 RNA in dermal fibroblast cells from patients with different progeroid syndromes using specific antisense oligonucleotides (ASOs) restored heterochromatin histone 3 lysine 9 and histone 3 lysine 27 trimethylation marks, reversed DNA methylation age, and counteracted the expression of senescence-associated secretory phenotype genes such as p16, p21, activating transcription factor 3 (ATF3), matrix metallopeptidase 13 (MMP13), interleukin 1a (IL1a), BTG anti-proliferation factor 2 (BTG2), and growth arrest and DNA damage inducible beta (GADD45b). Moreover, systemic delivery of ASOs rescued the histophysiology of tissues and increased the life span of a Hutchinson-Gilford progeria syndrome mouse model. Transcriptional profiling of human and mouse samples after L1 RNA depletion demonstrated that pathways associated with nuclear chromatin organization, cell proliferation, and transcription regulation were enriched. Similarly, pathways associated with aging, inflammatory response, innate immune response, and DNA damage were down-regulated. Our results highlight the role of L1 RNA in heterochromatin homeostasis in progeroid syndromes and identify a possible therapeutic approach to treat premature aging and related syndromes.