siRNA Oligonucleotides Research Articles

Abstract 3038: IDH1 facilitates melanoma cell survival under metabolic stress

Abstract Introduction: Novel immunotherapies and targeted therapies have generated remarkable responses for patients with melanoma relative to historic treatments, but these responses are only seen in about 50% of patients. This suggests that treatment resistance is an ongoing challenge for a significant number of patients with melanoma, leaving an urgent need for improved therapeutic strategies. In our previous studies, we demonstrated that oxidative stress has an important role in drug resistance, and that wild type isocitrate dehydrogenase 1 (IDH1) is a major source of cytosolic NADPH that maintains redox homeostasis of cells under hypoxic and metabolic stress. This critical enzyme has not been thoroughly evaluated in melanoma. Herein, we explored the expression and function of IDH1 in human melanoma and the role of IDH1 in regulating melanoma metabolism. Methods: We evaluated IDH1 expression in primary and metastatic melanoma using The Cancer Genome Atlas (TCGA). We performed IDH1 knockdown by siRNA oligos, and evaluated cell viability by Trypan blue and PicoGreen assays under normal and nutrient-deprived conditions. Cellular reactive oxygen species (ROS) levels were determined by the DCFDA method. Metastatic activity of these cells was measured by transwell migration assays. In order to determine the impact of IDH1 in cellular metabolism, metabolomics profiling was performed by using LC-MS. These experiments were performed in A375 and SK-Mel 28 cell lines. Results: Analysis of TCGA data from melanoma samples showed IDH1 is highly overexpressed in primary and metastatic melanoma, and higher levels are associated with decreased progression-free survival in patients. Further, we validated that IDH1 is overexpressed in tumors by comparing normal skin tissue versus tumor samples by IHC and protein expression arrays. Silencing IDH1 impaired cell proliferation and migration (vs. control) in a nutrient-deprived microenvironment, but this effect was not seen in nutrient abundance. Metabolomics revealed that inhibiting IDH1 significantly decreased NADPH, α-ketoglutarate (αKG), and GSH levels with a corresponding increase in ROS levels and an impairment of mitochondrial function. In addition, silencing IDH1 sensitized melanoma cells to temozolomide (TMZ), a DNA-alkylating agent, as indicated by a decrease in relative cell survival compared with controls. Conclusion: IDH1 plays a critical role in tumorigenesis and chemoresistance in melanoma. Our data show that IDH1 inhibition sensitizes melanoma cells to TMZ therapy. Our study suggests that IDH1 is a potential target in melanoma as a monotherapy or in combination with existing chemotherapies. Citation Format: Mehrdad Zarei, Ali Vaziri-Gohar, Jonathan Hue, Omid Hajihassani, Erryk Katayama, Hallie Graor, Jordan Winter, Luke Rothermel. IDH1 facilitates melanoma cell survival under metabolic stress [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3038.

Abstract 101: JAK-independent STAT5 signaling as a node of therapeutic vulnerability in glioblastoma

Abstract Glioblastoma (GBM), the most common primary brain tumor in adults, comprises all WHO Grade IV gliomas of astrocytic origin. Despite extensive characterization of the molecular and genetic features of GBM, there remains a paucity of viable treatment options, and this disease confers a dismal 5% 5-year survival rate. Genomic amplification of epidermal growth factor receptor (EGFR) occurs in 40-60% of primary GBM. Half of all EGFR-amplified cases of GBM also harbor EGFRvIII, a constitutively active, truncated variant of EGFR. EGFR and EGFRvIII transduce oncogenic signals via cytoplasmic mediators, including the JAK/STAT signal transduction pathway. The oncogenic role of STAT3 has been well-studied in CNS malignancies; STAT3 has been found to induce the expression of target genes involved in the proliferative and invasive phenotypes of GBM. However, little is known about the mechanism and role of STAT5 activation in GBM progression. Microenvironmental cues and EGFRvIII signaling, lost in adherent cell culture, are required for STAT5 activation, making STAT5 difficult to study by conventional cell culture methods. Our recent data from patient-derived xenograft (PDX) cultures demonstrates that activation of STAT5 is important in GBM cell invasion and survival. Further, STAT5 activation is present predominantly at the invasive periphery of the tumor. In this study, we seek to characterize the mechanism and role of STAT5 activation downstream of EGFRvIII in GBM. RNA sequencing of PDX tumors expressing stable shRNA knockdown of STAT3 or STAT5 indicate both redundant and distinct transcriptional changes induced by STAT3 and STAT5 in GBM. We also find that while STAT3 activation is dependent upon JAK1/2, STAT5 activation is JAK-independent downstream of EGFRvIII in GBM. Depletion of JAK1/2 expression by siRNA oligonucleotides and pharmacologic inhibition of JAK1/2 suppressed STAT3 activation but not STAT5. In fact, targeting JAK2 with kinase inhibitor WP1066 suppressed STAT3 activation, but increased activation of STAT5 in GBM cells expressing EGFR/EGFRvIII. Inhibition of Src family kinases (SFK), a family of upstream signaling effectors of the JAK/STAT pathway, attenuates both STAT3 and STAT5 activation. These results suggest that activation of STAT5 may provide a therapeutic escape mechanism to JAK-STAT3 inhibition in GBM, necessitating dual STAT3 and STAT5 inhibition to successfully inhibit the distinct oncogenic signals transmitted by STAT3 and STAT5 to promote GBM cell survival and resistance. Citation Format: Mylan Blomquist, Serdar Tuncali, Christopher Sereduk, Jean Kloss, Joseph Loftus, Nhan Tran. JAK-independent STAT5 signaling as a node of therapeutic vulnerability in glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 101.

Evaluation of 3′-phosphate as a transient protecting group for controlled enzymatic synthesis of DNA and XNA oligonucleotides

Chemically modified oligonucleotides have advanced as important therapeutic tools as reflected by the recent advent of mRNA vaccines and the FDA-approval of various siRNA and antisense oligonucleotides. These sequences are typically accessed by solid-phase synthesis which despite numerous advantages is restricted to short sequences and displays a limited tolerance to functional groups. Controlled enzymatic synthesis is an emerging alternative synthetic methodology that circumvents the limitations of traditional solid-phase synthesis. So far, most approaches strived to improve controlled enzymatic synthesis of canonical DNA and no potential routes to access xenonucleic acids (XNAs) have been reported. In this context, we have investigated the possibility of using phosphate as a transient protecting group for controlled enzymatic synthesis of DNA and locked nucleic acid (LNA) oligonucleotides. Phosphate is ubiquitously employed in natural systems and we demonstrate that this group displays most characteristics required for controlled enzymatic synthesis. We have devised robust synthetic pathways leading to these challenging compounds and we have discovered a hitherto unknown phosphatase activity of various DNA polymerases. These findings open up directions for the design of protected DNA and XNA nucleoside triphosphates for controlled enzymatic synthesis of chemically modified nucleic acids.

Silencing of TP73-AS1 impairs prostate cancer cell proliferation and induces apoptosis via regulation of TP73.

Prostate cancer is a malignant disease that severely affects thehealth and comfort of themale population. The long non-coding RNA TP73-AS1 has been shown to be involved in the malignant transformation of various human cancers. However, whether TP73-AS1 contributes toprostate cancer progression has not been reported yet. Accordingly, here weaimed to report the role of TP73-AS1 in the development and progression of prostate cancer and determine its relationship with TP73. TP73-AS1-specific siRNA oligo duplexes were used to silence TP73-AS1 in DU-145 and PC-3 cells. Results indicated that TP73-AS1 was upregulated whereas TP73 was downregulated in prostate cancer cells compared to normal prostate cells and there was a negative correlation between them. Besides, loss of function experiments of TP73-AS1 in prostate cancer cells strongly induced cellular apoptosis, interfered with the cell cycle progression, and modulated related pro- and anti-apoptotic gene expression. Colony formation and migration capacities of TP73-AS1-silenced prostate cancer cells were also found to be dramatically reduced. Our findings provide novel evidence that suggests a chief regulatory role for the TP73-TP73-AS1 axis in prostate cancer development and progression, suggesting that the TP73/TP73-AS1 axis can be a promising diagnostic and therapeutic target for prostate cancer.

Cyclic peptides nanospheres: A ‘2-in-1′ self-assembled delivery system for targeting nucleus and cytoplasm

Vascular endothelial growth factor (VEGF) is considered as one of the vital growth factors for angiogenesis, which is primarily responsible for the progress and maintenance of new vascular network in tumor. Numerous studies report that inhibition of VEGF-induced angiogenesis is a potent technique for cancer suppression. Recently, RNA interference, especially small interfering RNA (siRNA) signified a promising approach to suppress the gene expression. However, the clinical implementation of biological macromolecules such as siRNA is significantly limited because of stability and bioavailability issues. Herein, self-assembled peptide nanospheres have been generated from L,L-cyclic peptides using hydrophobic (Trp), positively charged (Arg) and cysteine (Cys) amino acid residues and demonstrated as vehicles for intracellular delivery of VEGF siRNA and VEGF antisense oligonucleotide. Formation of peptide nanostructures is confirmed by HR-TEM, AFM, SEM and DLS analysis. Possible mechanism of self-assembly of the cyclic peptides and their binding with macromolecules are demonstrated by in-silico analysis. Gel electrophoresis reveals that the newly generated peptide based organic materials exhibit strong binding affinity toward siRNAs / antisense oligonucleotides (ASOs) at optimum concentration. Flow cytometry and confocal microscopy results confirm the efficiency of the new biomaterials toward the intracellular delivery of fluorescent labeled siRNA / ASOs. Furthermore, VEGF expression evaluated by western blot and RT-PCR upon the delivery of functional VEGF siRNA/ASOs suggests that very low concentrations of VEGF siRNA/ASOs cause significant gene knockdown at protein and mRNA levels, respectively.

Role of alternatively spliced, pro-survival Protein Kinase C delta VIII (PKCδVIII) in ovarian cancer.

Ovarian cancer is the deadliest malignant disease in women. Protein Kinase C delta (PRKCD; PKCδ) is serine/threonine kinase extensively linked to various cancers. In humans, PKCδ is alternatively spliced to PKCδI and PKCδVIII. However, the specific function of PKCδ splice variants in ovarian cancer has not been elucidated yet. Hence, we evaluated their expression in human ovarian cancer cell lines (OCC): SKOV3 and TOV112D, along with the normal T80 ovarian cells. Our results demonstrate a marked increase in PKCδVIII in OCC compared to normal ovarian cells. Therefore, we elucidated the role of PKCδVIII and the underlying mechanism of its expression in OCC. Using overexpression and knockdown studies, we demonstrate that PKCδVIII increases cellular survival and migration in OCC. Further, overexpression of PKCδVIII in T80 cells resulted in increased expression of Bcl2 and knockdown of PKCδVIII in OCC decreased Bcl2 expression. Using co‐immunoprecipitations and immunocytochemistry, we demonstrate nuclear localization of PKCδVIII in OCC and further show increased association of PKCδVIII with Bcl2 and Bcl‐xL in OCC. Using PKCδ splicing minigene, mutagenesis, siRNA and antisense oligonucleotides, we demonstrate that increased levels of alternatively spliced PKCδVIII in OCC is regulated by splice factor SRSF2. Finally, we verified that PKCδVIII levels are elevated in samples of human ovarian cancer tissue. The data presented here demonstrate that the alternatively spliced, signaling kinase PKCδVIII is a viable target to develop therapeutics to combat progression of ovarian cancer.

Abstract 11173: Long Non-Coding RNA Linc00667 Modulates Cardiac Sodium Channel Downregulation During Ischemia

Background: The voltage-gated cardiac sodium channel α subunit ( SCN5A ; encoding Na v 1.5) plays a key role in cardiac conduction. The sodium channel is downregulated in cardiomyopathy, contributing to arrhythmic risk. In part, this downregulation is because of abnormal mRNA splicing and reduced mRNA stability. We recently reported that microRNA (miRNA)-448 contributes to the SCN5A mRNA instability in ischemic conditions. Objective: Long intergenic non-protein coding RNA 667 (LINC00667) is expressed in heart and can bind miRNA-448. We investigated the interaction of these two non-coding RNAs (ncRNAs) in regulating SCN5A . Methods: The expressions of SCN5A , miR-448 and LINC00667 were determined by quantitative real time polymerase chain reaction. The effects of ncRNAs on sodium current were determined in induced pluripotent stem cells-derived cardiomyocytes. The interaction between two ncRNAs was confirmed by pull-down assay. Results: LINC00667 has two binding sites for miR-448. A pull-down assay using biotin-tagged miR-448 showed direct binding of LINC00667 to miR-448. This indicates that LINC00667 could act as a competitor through the sponge function to miR-448. The expression of LINC00667 was decreased in human ischemic cardiomyopathy tissues compared to healthy controls. DFX treatment, a hypoxia mimetic chemical, or hypoxic condition (2% O 2 ) resulted in reduced LINC00667 and reduced SCN5A mRNA in cardiomyocyte. Furthermore, LINC00667 inhibition by siRNA or antisense oligo showed reduction of SCN5A mRNA. This reduction is expected to enhance the effect of miR-448 and is expected to be inhibited by treatment with a DNA construct containing the miR-448 binding site of LINC00667 or "miR-448 sponge". Conclusion: SCN5A is downregulated by hypoxia. In addition to upregulation of miR-448, which destabilizes SCN5A mRNA by direct binding, LINC00667 inhibition of miR-448 was relieved by hypoxia increasing the effect of miR-448 on SCN5A . Therefore, LINC00667 upregulation may represent a method to decrease arrhythmic risk during cardiomyopathy by raising SCN5A levels.

ETS1, ELK1, and ETV4 Transcription Factors Regulate Angiopoietin-1 Signaling and the Angiogenic Response in Endothelial Cells.

BackgroundAngiopoietin-1 (Ang-1) is the main ligand of Tie-2 receptors. It promotes endothelial cell (EC) survival, migration, and differentiation. Little is known about the transcription factors (TFs) in ECs that are downstream from Tie-2 receptors.ObjectiveThe main objective of this study is to identify the roles of the ETS family of TFs in Ang-1 signaling and the angiogenic response.MethodsIn silico enrichment analyses that were designed to predict TF binding sites of the promotors of eighty-six Ang-1-upregulated genes showed significant enrichment of ETS1, ELK1, and ETV4 binding sites in ECs. Human umbilical vein endothelial cells (HUVECs) were exposed for different time periods to recombinant Ang-1 protein and mRNA levels of ETS1, ELK1, and ETV4 were measured with qPCR and intracellular localization of these transcription factors was assessed with immunofluorescence. Electrophoretic mobility shift assays and reporter assays were used to assess activation of ETS1, ELK1, and ETV4 in response to Ang-1 exposure. The functional roles of these TFs in Ang-1-induced endothelial cell survival, migration, differentiation, and gene regulation were evaluated by using a loss-of-function approach (transfection with siRNA oligos).ResultsAng-1 exposure increased ETS1 mRNA levels but had no effect on ELK1 or ETV4 levels. Immunostaining revealed that in control ECs, ETS1 has nuclear localization whereas ELK1 and ETV4 are localized to the nucleus and the cytosol. Ang-1 exposure increased nuclear intensity of ETS1 protein and enhanced nuclear mobilization of ELK1 and ETV4. Selective siRNA knockdown of ETS1, ELK1, and ETV4 showed that these TFs are required for Ang-1-induced EC survival and differentiation of cells, while ETS1 and ETV4 are required for Ang-1-induced EC migration. Moreover, ETS1, ELK1, and ETV4 knockdown inhibited Ang-1-induced upregulation of thirteen, eight, and nine pro-angiogenesis genes, respectively.ConclusionWe conclude that ETS1, ELK1, and ETV4 transcription factors play significant angiogenic roles in Ang-1 signaling in ECs.

Depletion of Branched-Chain Ketoacid Dehydrogenase Kinase (BDK) Upregulates Insulin-Stimulated Glucose Uptake in Muscle Cells

Abstract Objectives Plasma levels of branched-chain amino acids (BCAAs) and their metabolites, branched-chain ketoacids (BCKAs) are increased in insulin resistance, a condition that can lead to type 2 diabetes mellitus (T2DM). BCAA catabolic enzymes are downregulated in diabetes and obesity. We previously showed that leucine and KIC suppressed insulin-stimulated glucose uptake in L6 myotubes. We have also shown that knocking down branched-chain ketoacid dehydrogenase (BCKD), an enzyme that decarboxylates BCKAs, suppressed insulin-stimulated glucose uptake. The objective of this study is to analyze how stimulating BCAA catabolic flux, by depleting branched-chain ketoacid dehydrogenase kinase (BDK), a negative regulator of BCKD, affects insulin sensitivity. We hypothesize that upregulating BCAA catabolism will increase insulin-stimulated glucose transport and attenuate insulin resistance. Methods L6 myoblasts were cultured in differentiation media for 4 days. On day 4 of differentiation, cells were transfected with control (SCR) or branched-chain ketoacid dehydrogenase kinase (BDK) siRNA oligonucleotides. Forty-eight hours later, myotubes were starved of serum- and amino acids for 3 hours then supplemented with or without KIC (200 mM) for 30 minutes. After, cells were incubated with or without insulin (100 nM) for 20 minutes. They were then harvested for immunoblotting or used for glucose transport assay. Results There was a 32% increase in insulin-stimulated glucose uptake with BDK depletion. KIC suppressed insulin-stimulated glucose uptake by 25% in control (SCR) cells; this suppression was attenuated in cells depleted of BDK. BDK depletion also reduced KIC-induced IRS-1Ser612 phosphorylation by 64% but had no effect on AktSer473 phosphorylation. Conclusions BDK depletion increased insulin-stimulated glucose transport, and attenuated KIC-induced suppression of insulin-stimulated glucose uptake, suggesting that increasing BCKD activity can be a therapeutic strategy against insulin resistance. Funding Sources Natural Science and Research Council (NSERC)

P041 CDKN2B-AS1 (ANRIL) expression is decreased in Inflammatory Bowel Disease epithelia and in Celiac, and its reduction is linked with induced cells proliferation

Abstract Background Long non-coding RNAs (lncRNAs) have attenuated expression in several immune-mediated disorders. Using mRNAseq of intestinal biopsies, we identified a widespread dysregulation of 459 lncRNAs in the ileum of treatment-naïve pediatric Crohn Disease (CD) patients. We noted that large fraction of downregulated lncRNAs were correlated with epithelial functions. CDKN2B-AS1 (ANRIL) is one of the top most down-regulated lncRNA in CD, and showed decreased expression in Ulcerative Colitis (UC) colon tissues in recent studies. Methods Transcriptomics data is used to evaluate CDKN2B-AS1 expression in mucosal biopsies datasets and in gut epithelia. siRNA oligonucleotides targeting most CDKN2B-AS1 transcripts using Lipofectamine RNAiMAX is used to modulate CDKN2B-AS1 function, and RT-PCR to evaluate mRNA expression. xCELLigence system with gold microelectrodes in plate base is used to measure the impedance as an indicator for cell index. Results CDKN2B-AS1 is down-regulated in bulk mucosal biopsies obtained from CD ileum [fold change (FC) -8, corrected p=1.6E-5], in UC rectum (FC -9.4, corrected p=3.9E-18), and in celiac duodenum (FC -2.3, corrected p=0.03) in comparison to controls. CDKN2B-AS1 is detected under basal conditions in HT-29 cells. Two sets of CDKN2B-AS1 siRNA oligonucleotides (targeting most transcripts) achieved up to 60% reduction in CDKN2B-AS1 expression in HT-29 cells (p=1E-04) as confirmed by RT-PCR using two sets of primers. CDKN2B-AS1 reduction significantly increased cell index as measured by xCELLigence (doubled the index, p=1.7E-03). mRNAseq of the CDKN2B-AS1 reduced HT-29, showed reduction of the tumor suppressor gene APC in CDKN2B-AS1 siRNA treated cells (FC=-1.75, p=0.04) and of TGFBR2 (FC=-1.75, p=0.04). In contrast, reduction of CDKN2B-AS1 resulted in increase in WNT11 (FC=2.1, p=0.02) and TGFB1 (FC=1.9, p=0.04), and induction of the replication associated topoisomerase TOP1MT (FC=2.7, p=0.001). We confirmed these mRNAseq results using RT-PCR. Finally, mimicking inflammation by treating HT-29 with IL1β and LPS, reduced CDKN2B-AS1 expression (by 70%, p=6.3E-04) and doubled the cell index (p=1.8E-03). Conclusion We detected reduced CDKN2B-AS1 expression in three inflammatory disease affecting the gut in different location along the GI tract. Using HT-29 model system we were able to show, as was previously shown, an increase in cell index in CDKN2B-AS1 siRNA treated cells. We supplement those showing effect on down-stream genes that may be relevant in controlling cell proliferation. We further show that upon inflammatory triggering of HT-29 cells, CDKN2B-AS1 expression is reduced and cell index is increased, which may suggest a potential role in epithelial renewal in inflammation.

Sodium bicarbonate transporter NBCe1 regulates proliferation and viability of human prostate cancer cells LNCaP and PC3.

Studies on cultured cancer cells or cell lines have revealed multiple acid extrusion mechanisms and their involvement in cancer cell growth and progression. In the present study, the role of the sodium bicarbonate transporters (NBCs) in prostate cancer cell proliferation and viability was examined. qPCR revealed heterogeneous expression of five NBC isoforms in human prostate cancer cell lines LNCaP, PC3, 22RV1, C4-2, DU145, and the prostate cell line RWPE-1. In fluorescence pH measurement of LNCaP cells, which predominantly express NBCe1, Na+ and HCO3–-mediated acid extrusion was identified by bath ion replacement and sensitivity to the NBC inhibitor S0859. NBCe1 knockdown using siRNA oligonucleotides decreased the number of viable cells, and pharmacological inhibition with S0859 (50 µM) resulted in a similar decrease. NBCe1 knockdown and inhibition also increased cell death, but this effect was small and slow. In PC3 cells, which express all NBC isoforms, NBCe1 knockdown decreased viable cell number and increased cell death. The effects of NBCe1 knockdown were comparable to those by S0859, indicating that NBCe1 among NBCs primarily contributes to PC3 cell proliferation and viability. S0859 inhibition also decreased the formation of cell spheres in 3D cultures. Immunohistochemistry of human prostate cancer tissue microarrays revealed NBCe1 localization to the glandular epithelial cells in prostate tissue and robust expression in acinar and duct adenocarcinoma. In conclusion, our study demonstrates that NBCe1 regulates acid extrusion in prostate cancer cells and inhibiting or abolishing this transporter decreases cancer cell proliferation.

Chemical Manipulation of the Endosome Trafficking Machinery: Implications for Oligonucleotide Delivery.

Antisense oligonucleotides (ASOs), siRNA and splice switching oligonucleotides (SSOs) all have immense potential as therapeutic agents, potential that is now being validated as oligonucleotides enter the clinic. However, progress in oligonucleotide-based therapeutics has been limited by the difficulty in delivering these complex molecules to their sites of action in the cytosol or nucleus of cells within specific tissues. There are two aspects to the delivery problem. The first is that most types of oligonucleotides have poor uptake into non-hepatic tissues. The second is that much of the oligonucleotide that is taken up by cells is entrapped in endosomes where it is pharmacologically inert. It has become increasingly recognized that endosomal trapping is a key constraint on oligonucleotide therapeutics. Thus, many approaches have been devised to address this problem, primarily ones based on various nanoparticle technologies. However, recently an alternative approach has emerged that employs small molecules to manipulate intracellular trafficking processes so as to enhance oligonucleotide actions. This review presents the current status of this chemical biology approach to oligonucleotide delivery and seeks to point out possible paths for future development.

Therapeutic targeting of STAT3 with small interference RNAs and antisense oligonucleotides embedded exosomes in liver fibrosis.

Hepatic fibrosis is a wound healing response that results in excessive extracellular matrix (ECM) accumulation in response to chronic hepatic injury. Signal transducer and activator of transcription 3 (STAT3) is an important transcription factor associated with the pathogenesis of liver fibrosis. Though a promising potential therapeutic target, there are no specific drug candidates for STAT3. Exosomes are extracellular vesicles generated by all cell types with a capacity to efficiently enter cells across different biological barriers. Here, we utilize exosomes as delivery conduit to specifically target STAT3 in liver fibrosis. Exosomes derived from clinical grade fibroblast-like mesenchymal stem cells (MSCs) were engineered to carry siRNA or antisense oligonucleotide (ASO) targeting STAT3 (iExosiRNA-STAT3 or iExomASO-STAT3 ). Compared to scrambled siRNA control, siRNA-STAT3, or ASO-STAT3, iExosiRNA-STAT3 or iExomASO-STAT3 showed enhanced STAT3 targeting efficiency. iExosiRNA-STAT3 or iExomASO-STAT3 treatments suppressed STAT3 levels and ECM deposition in established liver fibrosis in mice, and significantly improved liver function. iExomASO-Stat3 restored liver function more efficiently when compared to iExosiRNA-STAT3 . Our results identify a novel anti-fibrotic approach for direct targeting of STAT3 with exosomes with immediate translational potential.

Delivery of Oligonucleotides Using a Self-Degradable Lipid-Like Material.

The world-first success of lipid nanoparticle (LNP)-based siRNA therapeutics (ONPATTRO®) promises to accelerate developments in siRNA therapeutics/gene therapy using LNP-type drug delivery systems (DDS). In this study, we explore the optimal composition of an LNP containing a self-degradable material (ssPalmO-Phe) for the delivery of oligonucleotides. siRNA or antisense oligonucleotides (ASO) were encapsulated in LNP with different lipid compositions. The hepatic knockdown efficiency of the target genes and liver toxicity were evaluated. The optimal compositions for the siRNA were different from those for ASO, and different from those for mRNA that were reported in a previous study. Extracellular stability, endosomal escape and cellular uptake appear to be the key processes for the successful delivery of mRNA, siRNA and ASO, respectively. Moreover, the compositions of the LNPs likely contribute to their toxicity. The lipid composition of the LNP needs to be optimized depending on the type of nucleic acids under consideration if the applications of LNPs are to be further expanded.

Site-Selective Artificial Ribonucleases: Renaissance of Oligonucleotide Conjugates for Irreversible Cleavage of RNA Sequences.

RNA-targeting therapeutics require highly efficient sequence-specific devices capable of RNA irreversible degradation in vivo. The most developed methods of sequence-specific RNA cleavage, such as siRNA or antisense oligonucleotides (ASO), are currently based on recruitment of either intracellular multi-protein complexes or enzymes, leaving alternative approaches (e.g., ribozymes and DNAzymes) far behind. Recently, site-selective artificial ribonucleases combining the oligonucleotide recognition motifs (or their structural analogues) and catalytically active groups in a single molecular scaffold have been proven to be a great competitor to siRNA and ASO. Using the most efficient catalytic groups, utilising both metal ion-dependent (Cu(II)-2,9-dimethylphenanthroline) and metal ion-free (Tris(2-aminobenzimidazole)) on the one hand and PNA as an RNA recognising oligonucleotide on the other, allowed site-selective artificial RNases to be created with half-lives of 0.5–1 h. Artificial RNases based on the catalytic peptide [(ArgLeu)2Gly]2 were able to take progress a step further by demonstrating an ability to cleave miRNA-21 in tumour cells and provide a significant reduction of tumour growth in mice.

A 2020 update on the progress of treatment and new drug clinical trials for hepatitis B

Long term antiviral therapy with nucleos(ti)ide analogues could suppress HBV viral load thereby prevent the progression to cirrhosis and hepatocellular carcinoma. Interferon-based therapy could result in sustained virological response in a fair proportion of patients and even HBsAg loss in a small proportion of them. Novel therapies aiming at functional cure (loss of HBsAg) are under active development. Among the categories of many, HBV core protein inhibitors are safe and could suppress the HBV DNA and HBV RNA, but only with modest effect on the level of HBsAg; silencing of HBV mRNA by siRNA or antisense oligonucleotides could produce meaningful and sustainable declining in HBsAg levels; immune modulators with different mode of action showed modest effect on the reduction of HBsAg, but with noticeable adverse event (especially transaminase flares) related to the mode of action. Novel clinical trial design on the combination or sequential use of innovative molecules will ultimately lead to the functional cure of CHB in the near future.

Ginkgo biloba leaf extract prevents diabetic nephropathy through the suppression of tissue transglutaminase.

The present study aimed to investigate the preventive effects of Ginkgo biloba leaf extract (GBE) against extracellular matrix (ECM) accumulation in a streptozotocin (STZ)-induced rat model of diabetic nephropathy (DN), and to determine its underlying molecular mechanism. In vivo, a rat model of DN was established by intraperitoneal injection of STZ, and the rats were subsequently administered GBE. The results demonstrated that GBE significantly decreased blood glucose, the urine protein excretion rate and ECM accumulation in DN rats. In addition, the development of DN significantly induced tissue transglutaminase (tTG) protein expression, which was detected by immunohistochemistry, western blotting and PCR analyses, while GBE administration decreased tTG expression in the diabetic kidney. In vitro, rat glomerular mesangial cells (HBZY-1 cells) cultured with high glucose were also treated with GBE. The concentrations of tTG, fibronectin, type IV collagen, transforming growth factor (TGF)-β and connective tissue growth factor (CTGF) were detected via ELISA. The results demonstrated that GBE notably decreased the concentration of these proteins, and tTG expression was positively associated with TGF-β. GBE also suppressed tTG expression of high glucose-treated HBZY-1 cells in a concentration-dependent manner. Furthermore, tTG protein expression was detected in high glucose-treated HBZY-1 cells transfected with small interfering RNA (siRNA) oligonucleotides against TGF-β and CTGF to investigate a possible mechanism of GBE-mediated inhibition of tTG. The results demonstrated that the tTG levels remained unchanged in CTGF siRNA-transfected cells, but were decreased in the GBE + CTGF siRNA group compared with the control siRNA group, suggesting that tTG may not be regulated by CTGF, and the inhibitory effect of GBE on tTG may not be associated with the direct inhibition of CTGF. However, tTG expression was decreased following the transfection with TGF-β siRNA, in which levels of tTG were similar compared with both the GBE group and GBE + TGF-β siRNA group, indicating that tTG may be regulated by TGF-β, and that the GBE-induced repression of tTG expression may be associated with the downregulation of TGF-β. Taken together, the results of the present study suggest that GBE prevented ECM accumulation by suppressing tTG expression in DN, which was predominantly mediated by TGF-β.

PFRED: A computational platform for siRNA and antisense oligonucleotides design.

PFRED a software application for the design, analysis, and visualization of antisense oligonucleotides and siRNA is described. The software provides an intuitive user-interface for scientists to design a library of siRNA or antisense oligonucleotides that target a specific gene of interest. Moreover, the tool facilitates the incorporation of various design criteria that have been shown to be important for stability and potency. PFRED has been made available as an open-source project so the code can be easily modified to address the future needs of the oligonucleotide research community. A compiled version is available for downloading at https://github.com/pfred/pfred-gui/releases/tag/v1.0 as a java Jar file. The source code and the links for downloading the precompiled version can be found at https://github.com/pfred.

Growth hormone receptor disrupts glucose homeostasis via promoting and stabilizing retinol binding protein 4.

Rationale: The molecular mechanisms underlying the pathogenesis of systemic insulin resistance in type 2 diabetes remain elusive. Growth hormone receptor (GHR) deficiency has long been known to improved insulin sensitivity. However, whether hepatic GHR overexpression or activation is a cause of insulin resistance is still unknown. The aim of this study was to identify the new role of GHR in systemic insulin resistance and explore the underlying mechanism.Method: Different samples obtained from obese humans, ob/ob mice, db/db mice, high-fat diet (HFD)-fed mice and primary mouse hepatocytes were used to evaluate the correlations between GHR and metabolic disorders. Recombinant adeno-associated viruses encoding GHR and STAT5 and GHR knockout mice were used to investigate the roles of hepatic GHR in glucose homeostasis. Tissue H&E, Oil Red O and PAS staining were performed for histomorphological analysis. Gel filtration chromatography was employed for the separation of serum RBP4-TTR complexes. Plasmids (related to GHR, STAT5 and HIF1α), siRNA oligos (siGHR and siSTAT5), luciferase activity and ChIP assays were used to explore the potential mechanism of hepatic GHR.Results: Here, we found that hepatic GHR expression was elevated during metabolic disorder. Accordingly, hepatic GHR overexpression disrupted systemic glucose homeostasis by promoting gluconeogenesis and disturbing insulin responsiveness in the liver. Meanwhile, hepatic GHR overexpression promoted lipolysis in white adipose tissue and repressed glucose utilization in skeletal muscle by promoting the circulating level of RBP4, which contributed to impaired systemic insulin action. A mechanistic study revealed that hepatic GHR disrupted systemic insulin sensitivity by increasing RBP4 transcription by activating STAT5. Additionally, overexpression of hepatic GHR promoted TTR transcriptional levels by enhancing the expression of HIF1α, which not only increased the protein stability of RBP4 but also inhibited renal clearance of RBP4 in serum.Conclusions: Hepatic GHR overexpression and activation accelerated systemic insulin resistance by increasing hepatic RBP4 production and maintaining circulating RBP4 homeostasis. Our current study provides novel insights into the pathogenesis of type 2 diabetes and its associated metabolic complications.

Preparation of Lipid-Conjugated siRNA Oligonucleotides for Enhanced Gene Inhibition in Mammalian Cells.

Nucleic acid conjugates are promising drugs for treating gene-related diseases. Conjugating specific units like lipids, cell-penetrating peptides, polymers, antibodies, and aptamers either at the 3'- or 5'-termini of a siRNA duplex molecule has resulted in a plethora of siRNA bioconjugates with improved stabilities in bloodstream and better pharmacokinetic values than unmodified siRNAs. In this sense, lipid-siRNA conjugates have attracted a remarkable interest for their potential value in facilitating cellular uptake. In this chapter, we describe a series of protocols involving the synthesis of siRNA oligonucleotides carrying either neutral or cationic lipids at the 3'- and 5'-termini. The resulting lipid-siRNA conjugates are aimed to be used as exogenous effectors for inhibiting gene expression by RNA interference. A protocol for the formulation of lipid siRNA using sonication in the presence of serum is described yielding interesting transfection properties for cell culture without the use of transfecting agents.