siRNA Oligonucleotides Research Articles

Advanced Nanosystems for Clinical Translation

Advanced Nanosystems for Clinical Translation

Splicing factors: Insights into their regulatory network in alternative splicing in cancer.

More than 95% of all human genes are alternatively spliced after transcription, which enriches the diversity of proteins and regulates transcript and/or protein levels. The splicing isoforms produced from the same gene can manifest distinctly, even exerting opposite effects. Mounting evidence indicates that the alternative splicing (AS) mechanism is ubiquitous in various cancers and drives the generation and maintenance of various hallmarks of cancer, such as enhanced proliferation, inhibited apoptosis, invasion and metastasis, and angiogenesis. Splicing factors (SFs) play pivotal roles in the recognition of splice sites and the assembly of spliceosomes during AS. In this review, we mainly discuss the similarities and differences of SF domains, the details of SF function in AS, the effect of SF-driven pathological AS on different hallmarks of cancer, and the main drivers of SF expression level and subcellular localization. In addition, we briefly introduce the application prospects of targeted therapeutic strategies, including small-molecule inhibitors, siRNAs and splice-switching oligonucleotides (SSOs), from three perspectives (drivers, SFs and pathological AS). Finally, we share our insights into the potential direction of research on SF-centric AS-related regulatory networks.

Addressing cancer signal transduction pathways with antisense and siRNA oligonucleotides.

Signal transduction pathways play key roles in the initiation, progression and dissemination of cancer. Thus, signaling molecules are attractive targets for cancer therapeutics and enormous efforts have gone into the development of small molecule inhibitors of these pathways. However, regrettably, there has been only moderate progress to date, primarily in connection with the RAS signaling pathway. Oligonucleotide-based drugs potentially offer several advantages for addressing signaling pathways, including their exquisite selectivity and their ability to exploit both enzymatic and nonenzymatic targets. Nonetheless, there are problems inherent in the oligonucleotide approach, not the least being the challenge of effectively delivering these complex molecules to intracellular sites within tumors. This survey article will provide a selective review of recent studies where oligonucleotides were used to address cancer signaling and will discuss both positive aspects and limitations of those studies. This will be set in the context of an overview of various cancer signaling pathways and small molecule approaches to regulate those pathways. The survey will also evaluate the challenges and opportunities implicit in the oligonucleotide-based approach to cancer signaling and will point out several possibilities for future research.

Abstract A097: Genomic and transcriptomic characterization of RNA methyltransferases in breast cancer

Abstract Background: Methylations of RNA are catalyzed by RNA methyltransferases (RNMTs) which regulate the structure, stability, translation, and function of almost every major class of human RNA. To date, more than 50 human RNMTs have been identified. Previous studies revealed that mutation and dysregulation of several RNMTs is associated with various human diseases, notably developmental disorders and cancer. However, the genomic and transcriptomic alterations of RNMT genes as well as their functional roles in cancer initiation and progression remain poorly characterized. This study seeks to characterize genetic alterations of 58 RNMTs in human cancer with an enriched focus on breast cancer. Furthermore, we will determine the associations between recurrent genetic alterations and gene expression levels of each RNMT, clinicopathological features, disease-free survival and disparities in breast cancer. Methods: Comprehensive genomic and transcriptomic analyses of 58 RNMTs in more than 10,000 primary tumors was performed using TCGA and METABRIC datasets. Copy number and gene expression of RNMTs were assessed by clinicopathological features of breast cancer in approximate 2,000 METABRIC breast tumors with long-term clinical follow-up data. Loss-of-function analysis was performed to examine RNMT candidates with important roles in growth and viability of breast cancer cells. Additionally, one candidate RNMT, FTSJ3 (FtsJ RNA methyltransferase homolog 3), was further pursued in order to assess its impact on cancer cell phenotypes in a panel of breast cancer cell lines. Results: We identified that a subset of RNMT genes, including TRMT12, NSUN2, and FTSJ3, have high frequencies of genomic amplification in a spectrum of human cancers, particularly in breast cancer. Several RNMTs, notably FTSJ3 and EMG1, are highly amplified and over-expressed in breast cancers of African-American women compared with that of European-American women. Breast cancer has been classified into five intrinsic subtypes with distinct risks and underlying biology. We found that different subtypes of breast cancer had different patterns of copy number and expression of each RNMT. Furthermore, we revealed that FTSJ3 was associated with higher grade and advanced stage of breast cancer. A genome-wide loss-of-function shRNA screen in a large panel of tumor lines indicated that FTSJ3 was required for the survival of some tumor cell lines. FTSJ3 depletion using siRNA oligos caused apoptosis and suppressed breast cancer cell survival with minimal effect on normal-like breast cancer cell survival. Conclusion: We identified a subset of RNMTs, notably FTSJ3, that were significantly associated with cancer aggressiveness and poor prognosis. Loss-of-function analysis revealed that FTSJ3 had important roles in promoting breast cancer cell growth and survival. Our findings provide a framework for further study of the functional consequences of RNMT alterations in human cancer, and for developing therapies that target RNMTs in future. Citation Format: Morenci M. Manning, Yuanyuan Jiang, Rui Wang, Lanxin Liu, Madison Bonahoom, Shomita Rode, Zeng-Quan Yang. Genomic and transcriptomic characterization of RNA methyltransferases in breast cancer [abstract]. In: Proceedings of the Twelfth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2019 Sep 20-23; San Francisco, CA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2020;29(6 Suppl_2):Abstract nr A097.

Myofibrillar Protein Abundance and Anabolic Signaling in Myotubes Depleted of E1 Alpha Subunit of Branched-Chain Ketoacid Dehydrogenase Complex

Abstract Objectives Branched-chain amino acids (BCAAs) are essential amino acids that are crucial for skeletal muscle anabolism. Thus, alterations in their levels are associated with muscle atrophic diseases such as cancer, chronic inflammatory and neurological disorders. Others have linked impairments in BCAA metabolism to the development of insulin resistance and its sequelae. Compared to the effects of theses amino acids, much less is known on how impairment in BCAA catabolism affects skeletal muscle. BCAA catabolism starts with the reversible transamination by the mitochondrial enzyme branched-chain aminotransferase 2 (BCAT2). This is followed by the irreversible carboxylation, catalyzed by branched-chain ketoacid dehydrogenase (BCKD) complex. We have shown that BCAT2 and BCKD are essential for the differentiation of skeletal myoblasts into myotubes. Here, we investigated the effect of depletion of BCAT2 or of E1a subunit of BCKD in differentiated myotubes. Methods On day 4 of differentiation, L6 myotubes were transfected with the following siRNA oligonucleotides: scrambled (control), BCAT2, or E1a subunit of BCKD. Results Forty-eight hours after transfection, compared to control or BCAT2 siRNA group, we observed improved myotube structure in BCKD-depleted cells. BCKD depletion augmented myofibrillar protein levels: myosin heavy chain (MHC, 2-fold) and tropomyosin (4-fold), P < 0.05, n = 3. To further analyze the increase in myofibrillar protein content, we examined signaling through mTORC1 (mechanistic target of rapamycin complex 1), a vital complex necessary for skeletal muscle anabolism. BCKD depletion increased the phosphorylation of mTORC1 upstream activator AKT (52%, P < 0.05, n = 3), and of mTORC1 downstream substrates by 25%-86%, consistent with the increase in myofibrillar proteins. Finally, in myotubes treated with the catabolic cytokine (tumor necrosis factor-a), BCKD depletion tended to increase the abundance of tropomyosin (a myofibrillar protein). Conclusions We showed that depletion of BCKD enhanced myofibrillar protein content and anabolic signaling. If these data are confirmed in vivo, development of dietary and other interventions that target BCKD abundance or functions may promote muscle protein anabolism in individuals with muscle wasting conditions. Funding Sources MHRC, NSERC York U.

Abstract B32: Silencing of oncogenic KRAS by a mutant-favoring short interfering RNA

Abstract Oncogenic mutations in the KRAS gene are well-established drivers of cancer. Promising preclinical strategies including RNA interference (RNAi) have been developed to target oncogenic KRAS function, yet a clinically effective therapy to directly target KRAS remains to be achieved. While genetic knockdown of mutant KRAS (mKRAS) with RNAi is one promising approach, current methods are not mutant-selective, and thus they also decrease normal KRAS levels, raising concerns about normal cell toxicity. Here, we describe a custom short interfering RNA (siRNA) oligonucleotide (D1) designed to preferentially bind the most commonly occurring RAS missense mutations in codons 12 and 13. Our approach designed the siRNA to have three mismatches binding wild-type mRNA, but only two mismatches for the oncogenic mRNAs, thus having binding affinities more favorable for the mutant-specific sequences. Using NIH/3T3 mouse fibroblasts stably expressing human KRAS WT or mutant cDNA sequences and KRAS mutant lung cancer cell lines, we determined that D1 siRNA preferentially reduced mKRAS mRNAs versus the wild-type sequence at the transcriptional and protein levels. Phenotypically, D1 siRNA significantly reversed mKRAS-induced morphologic and growth transformation of NIH/3T3 cells. Furthermore, D1 siRNA significantly impaired the proliferation of several mKRAS lung cancer cells in 2D as well as 3D spheroids embedded in an extracellular matrix. To overcome clinical limitations of delivering the siRNA to lung tumors, we evaluated lipid nanoparticles (LNPs) as carriers of D1 siRNAs in orthotopic mKRAS lung cancer models and observed that Cy5.5-labeled LNPs are highly enriched in lung tumors and metastases. Compared with control siRNA-LNP treated mice, 344SQ (KRAS G12D) tumor-bearing mice injected once with D1 siRNA-LNPs had drastically decreased KRAS protein levels in tumors 72 h later (73% decrease, p=0.001). Using an orthotopically injected luciferase-labeled A549 (KRAS G12S) human lung cancer model, D1 siRNA-LNP treated mice had pronounced reductions in luciferase signal (56% reduction, p=0.045), chest wall metastases (67% reduction, p<0.001) and contralateral lung metastases (73% reduction, p<0.01). Interestingly, D1 siRNA-LNP did not impair tumor growth when applied in the 344SQ mKRAS orthotopic mouse lung model, consistent with the mKRAS-independent growth of this line. This result supports our conclusion that the effects of D1 siRNA-LNP in the A549 model are KRAS specific and highlight the importance of determining RAS-dependency a priori for KRAS-targeting therapeutics. Taken together, our data indicate a novel use of RNAi to specifically target oncogenic KRAS-driven cancers. Citation Format: Bjoern Papke, Amanda E.D. Van Swearingen, Anne Y. Feng, Salma H. Azam, Emily B. Harrison, Runying Yang, Adrienne D. Cox, Channing J. Der, Chad V. Pecot. Silencing of oncogenic KRAS by a mutant-favoring short interfering RNA [abstract]. In: Proceedings of the AACR Special Conference on Targeting RAS-Driven Cancers; 2018 Dec 9-12; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(5_Suppl):Abstract nr B32.

MicroRNA-374a-5p inhibits neuroinflammation in neonatal hypoxic-ischemic encephalopathy via regulating NLRP3 inflammasome targeted Smad6

AimsNeonatal hypoxic-ischemic encephalopathy (HIE) is still an important cause of neurological dysfunction. At present, there is no reliable biochemical index in clinical examination. Increasing evidence demonstrates that microRNAs (miRNAs) are involved in the process of HIE, and miR-374a-5p is down-regulated in HIE infants. In this study, the aim is to investigate the role and mechanism of miR-374a-5p in HIE. Main methodsSprague-Dawley (SD) rats were used to establish model of neonatal HIE, pathologic changes and inflammatory response of brain tissues were measured. Subsequently, primary microglia were induced by LPS (1 μg/ml) in vitro, the miR-374a-5p mimic, Ad-Smad6 adenovirus vector and NLRP3 siRNA oligo were applied for microglial transfection. Furthermore, the target relationship between miR-374a-5p and Smad6 was analyzed, while microglia activity and inflammatory factor (IL-1β, TNF-α and IL-6) levels were detected. Key findingsHerein, we found that over-expression of miR-374a-5p significantly attenuated brain injury and strongly inhibited the release of pro-inflammatory cytokines in neonatal rat HIE model. In vitro, miR-374a-5p inhibited LPS-induced microglial pro-inflammatory cytokines production by regulating NLRP3 inflammasome. In addition, Smad6 was identified as a direct target of miR-374a-5p, and miR-374a-5p had a negative regulatory effect on Smad6 expression. By targeting Smad6, miR-374a-5p inhibited the activation of NLRP3 inflammatory signals in microglia and the subsequent release of pro-inflammatory factors. SignificanceOur study recognized that miR-374a-5p as a novel regulator of microglial activation in neonatal HIE highlighted potential therapeutic target for the treatment of neonatal hypoxic-ischemic brain injury.

Renal miR‐195 mediates TNF‐dependent inhibition of NKCC2

We previously showed that tumor necrosis factor‐alpha (TNF) produced by renal epithelial cells inhibits Na+‐K+‐2Cl− cotransporter (NKCC2) activity as part of a mechanism that attenuates increases in blood pressure in response to elevated in sodium intake. However, the mechanisms by which TNF inhibits NKCC2 isoform expression and activity have not been studied. Previous miRNA array studies have found that miR‐195 is one of the most abundant microRNAs expressed in the kidney. In the present study, we demonstrate by qRT‐PCR that TNF up‐regulates mmu‐miR‐146a, ‐195, and ‐203 and down‐regulates mmu‐miR‐30c and ‐100 in the medullary thick ascending limb (mTAL). Most notably, qRT‐PCR analysis indicated that the level of miR‐195 was 4‐fold higher in mTAL tubules isolated from the renal medulla of male mice given a single intrarenal injection of murine recombinant TNF (5ng/g body weight) compared with the control group (saline injection). Data obtained using a TAL‐specific TNF knockout (KO) mouse model showed that miR‐195 abundance in the mTAL tubules from these mice was significantly decreased by 58% (P=0.04, n=4) compared with control mice (floxed TNF littermate controls) in response to ingestion of high salt (HS; 1% NaCl in drinking water) for 3 days. Concomitantly, NKCC2A isoform mRNA abundance was increased 3‐fold while phospho (p)NKCC2 expression increased approximately 64% in the renal medulla of the TAL‐specific TNF KO mice compared with littermate control mice. Collectively, the data suggest that TNF‐derived from the TAL in response to HS intake is an important inducer of miR‐195 levels. Using the prediction tool TargetScan v5.1, we identified a potential miR‐195 binding site in the 3′‐UTR of NKCC2A. Accordingly, intrarenal injection of miR‐195 inhibited the HS‐mediated increases in NKCC2A mRNA and concurrent pNKCC2 expression by 81% and 59%, respectively, in the renal medulla from TAL‐specific TNF KO mice. Consistent with the in vivo effects of TNF on miR‐195, treatment of primary cultures of mouse mTAL cells with 1 nM TNF for 4 hr increased miR‐195 expression more than 3‐fold (P=0.01; n=4). Moreover, NKCC2A mRNA and pNKCC2 expression was inhibited by 76% (P=0.02; n=4) and 61% (P=0.01; n=4), respectively, in mTAL cells exposed to hypertonic conditions (400 mOsmol/kg H2O) following cotreatment with miR‐195 and a short hairpin (sh)TNF silencing lentivirus construct. Silencing of TNFR1 in mTAL cells with a siRNA oligonucleotide inhibited the TNF‐mediated increase of miR‐195 expression and prevented the inhibitory effects of TNF by 73% (P=0.03; n=4), as well as suppressing the increase in pNKCC2 expression. These data identify miR‐195 as an important downstream target of TNFR1‐dependent signaling, which suppresses NKCC2A and pNKCC2 expression in mTAL cells. In summary, the present findings uncover a novel physiological mechanism in which miR‐195 induction via activation of TNFR1‐dependent signaling suppresses NKCC2 expression and activity, suggesting that miR‐195 expression may contribute to the regulation of salt and water excretion and blood pressure via modulation of NKCC2A isoform expression and activity.Support or Funding InformationThis work was supported by NIH grant R01HL133077

Orphan designation: Synthetic double-stranded siRNA oligonucleotide directed against delta-aminolevulinic acid synthase 1 mRNA, covalently linked to a ligand containing three N-acetylgalactosamine residues (givosiran), Treatment of acute hepatic porphyria

Orphan designation: Synthetic double-stranded siRNA oligonucleotide directed against delta-aminolevulinic acid synthase 1 mRNA, covalently linked to a ligand containing three N-acetylgalactosamine residues (givosiran), Treatment of acute hepatic porphyria

Orphan designation: Synthetic double-stranded siRNA oligonucleotide directed against SERPINA1 mRNA and containing four modified nucleosides which form a ligand cluster of four N-acetylgalactosamine residues, Treatment of congenital alpha-1 antitrypsin deficiency

Orphan designation: Synthetic double-stranded siRNA oligonucleotide directed against SERPINA1 mRNA and containing four modified nucleosides which form a ligand cluster of four N-acetylgalactosamine residues, Treatment of congenital alpha-1 antitrypsin deficiency

Calcium-Mediated In Vitro Transfection Technique of Oligonucleotides with Broad Chemical Modification Compatibility.

Oligonucleotide drugs (ODs) have gained increasing attention owing to their promising therapeutic potential. One major obstacle that ODs have been facing is the lack of appropriate in vitro validation systems that can predict in vivo activity and toxicity. We have devised a transfection method called CEM (Ca2+-enrichment method), where the simple enrichment of calcium ion with calcium chloride in culture medium potentiates the activity of various types of naked oligonucleotides including gapmers, siRNA, and phosphorodiamidate morpholino antisense oligonucleotides (PMO) in many cultured cell lines with limited cytotoxicity. We here describe a precise procedure of the method. Besides the benefit of the CEM's predictive power to accurately estimate in vivo activity of ODs of your interest in drug discovery and development settings, this cost-efficient, easy-to-access method can be a robust laboratory technique to modulate gene expressions with ODs with a variety of mechanisms of action.

Cholinergic α5 nicotinic receptor is involved in the proliferation and invasion of human prostate cancer cells.

Nicotinic acetylcholine receptor (nAChR) subunit α5 (α5-nAChR) is involved in tumor cell proliferation, inhibition of apoptosis, progression of metastasis, and induction of angiogenesis in certain solid tumors. However, the role of α5-nAChR in prostate cancer cell growth and metastasis is unclear. In the present study, the role of α5-nAChR in cell proliferation, migration, invasion and apoptosis was investigated by silencing the expression levels of α5-nAChR in the prostate cancer cell lines DU145 and PC3. A siRNA oligonucleotide targeting α5-nAChR was designed. The cell proliferation of DU145 and PC3 cell lines was analyzed by the Cell Counting Kit-8 (CCK-8) assay. Cell migratory and invasive activities were determined using wound healing and Transwell assays, respectively. Western blot analysis was used to quantify α5-nAChR, p-AKT and p-ERK1/2 levels in DU145 and PC3 cells. Knockdown of α5-nAChR was associated with decreased cell proliferation, migration, invasion and increased apoptosis. In addition, decreased phosphorylation levels of AKT and ERK1/2 were revealed following α5-nAChR knockdown in DU145 and PC3 cells compared with those observed in the scramble control samples. The expression levels of the apoptosis-related proteins were altered following silencing of α5-nAChR. In summary, the data indicated that α5-nAChR was involved in the proliferation and invasion of human prostate cancer cells.

39P - The regulation of INK4 locus by long non-coding RNAs

BackgroundINK4 locus is located on human chromosome 9p21 region and encodes three tumor suppressor genes, p15, p16 and ARF. So far, we revealed that a long non-coding RNA (lncRNA), ANRIL, transcribed from INK4 locus represses the transcription of p15 and p16 genes. ANRIL associates with polycomb protein complexes and recruits them on INK4 locus, leading to the transcriptional repression. The depletion of ANRIL inhibits the proliferation of cancer cells such as non-small cell lung cancer and colorectal cancer, indicating that ANRIL functions to promote cancer cells proliferation. Recently, we found a novel lncRNA induced by oncogenic Ras signal (named LION: lncRNA induced by oncogenic Ras signal). In this study, we showed that LION is involved in the transcriptional regulation of INK4 locus and cell proliferation. MethodsHuman non-small cell lung cancer cell H1299 and colorectal cancer cell HCT116 were transfected with siRNA oligonucleotides against LION. The expression analysis of INK4 locus genes was performed by quantitative reverse transcription-polymerase chain reaction (Q-RT-PCR). Cell cycle analysis was performed by using Muse cell analyzer. ResultsRT-PCR analysis showed that LION is highly expressed in several cancer cells such as non-small cell lung cancer, cervical cancer and colorectal cancer compared with normal lung fibroblasts. Silencing LION by siRNA oligonucleotides inhibits the proliferation of H1299, HCT116 and HeLa cells. Q-RT-PCR analysis showed that silencing LION increases the p15 and p16 mRNA, suggesting that LION is involved in the transcriptional repression of INK4 locus. Cell cycle analysis showed that silencing LION causes G2/M phase arrest in cell cycle, suggesting that LION functions to promote G2/M transition. ConclusionsLION is involved in the promotion of cancer cells proliferation such as H1299 and HCT116 cells via regulating p15, p16 and other genes related to G2/M phase control. Legal entity responsible for the studyThe authors. FundingJSPS KAKENHI. DisclosureAll authors have declared no conflicts of interest.

Abemaciclib Is Effective Against Pancreatic Cancer Cells and Synergizes with HuR and YAP1 Inhibition.

Mutation or promoter hypermethylation of CDKN2A is found in over 90% of pancreatic ductal adenocarcinomas (PDAC) and leads to loss of function of cell-cycle inhibitors p16 (INK4A) and p14 (ARF) resulting in unchecked proliferation. The CDK4/6 inhibitor, abemaciclib, has nanomolar IC50s in PDAC cell lines and decreases growth through inhibition of phospho-Rb (pRb), G1 cell-cycle arrest, apoptosis, and the senescent phenotype detected with β-galactosidase staining and relevant mRNA elevations. Daily abemaciclib treatments in mouse PDAC xenograft studies were safe and demonstrated a 3.2-fold decrease in tumor volume compared with no treatment (P < 0.0001) accompanying a decrease in both pRb and Ki67. We determined that inhibitors of HuR (ELAVL1), a prosurvival mRNA stability factor that regulates cyclin D1, and an inhibitor of Yes-Associated Protein 1 (YAP1), a pro-oncogenic, transcriptional coactivator important for CDK6 and cyclin D1, were both synergistic with abemaciclib. Accordingly, siRNA oligonucleotides targeted against HuR, YAP1, and their common target cyclin D1, validated the synergy studies. In addition, we have seen increased sensitivity to abemaciclib in a PDAC cell line that harbors a loss of the ELAVL1 gene via CRISP-Cas9 technology. As an in vitro model for resistance, we investigated the effects of long-term abemaciclib exposure. PDAC cells chronically cultured with abemaciclib displayed a reduction in cellular growth rates (GR) and coresistance to gemcitabine and 5-fluorouracil (5-FU), but not to HuR or YAP1 inhibitors as compared with no treatment controls. We believe that our data provide compelling preclinical evidence for an abemaciclib combination-based clinical trial in patients with PDAC. IMPLICATIONS: Our data suggest that abemaciclib may be therapeutically relevant for the treatment in PDAC, especially as part of a combination regimen inhibiting YAP1 or HuR.

Current approaches for treatment of hereditary transthyretin amyloidosis (ATTR)

Rare, progressive, and fatal is a short description for autosomal dominant hereditary transthyretin (TTR) amyloidosis (ATTR). In the absence of a family background, delays in diagnosis are common. ATTR is often represented by a progressive, axonal fiber-length-dependent polyneuropathy (motor, autonomic, sensory). Cardiovascular, gastrointestinal, renal and ocular manifestations are frequently observed. ATTR is caused by mutated serum protein transthyretin (TTRm), which results in amyloid deposits.The three current concepts of ATTR treatment aim to replace, stabilize and reduce TTR synthesis. (i) The mutant TTRm is almost completely replaced in the peripheral blood after orthotopic liver transplantation by the synthesis of the wild-type (TTRwt) in the donor liver. (ii) Low molecular weight compounds stabilize the TTR tetramer and thereby minimize the formation of amyloid precursors. (iii) Gene silencers (mRNA-inhibiting oligonucleotides) reduce liver-secreted TTRm and TTRwt.Liver transplantation (LTx) is an established procedure in ATTR patients. LTx significantly prolongs survival, especially in early stage patients (< 50 years) with variant ATTRV30M. Some non-ATTRV30M variants show a higher mortality after LTx. The progression of the disease can be slowed down but not prevented by LTx. Diflunisal is a low-cost, off-label drug from the NSAID group. Similar to Tafamidis, it stabilizes the TTR tetramer. Tafamidis has been approved for the treatment of stage 1 ATTR since 2011. It is administered orally and used in patients with polyneuropathy and more recently with cardiomyopathy. Inotersen represents an antisense oligonucleotide that is administered subcutaneously (s. c.) once a week. Patisiran acts as a siRNA oligonucleotide administered intravenously (i. v.) every three weeks in combination with premedication. Both gene silencers were approved in 2018 for the treatment of ATTR stages 1 and 2. The new era of TTR gene silencers now affords an update of algorithms used for treatment of ATTR.

Fowl Adenovirus Serotype 4 Influences Arginine Metabolism to Benefit Replication.

Hydropericardium syndrome (HPS) is caused by fowl adenovirus serotype 4 (FAdV-4). HPS has caused outbreaks in Chinese populations of broiler chickens since 2015. However, little is known about the molecular mechanisms underlying HPS. In this study, we used transcriptomic analysis to screen differentially expressed genes (DEGs) in the livers of FAdV-4-infected and noninfected chicks. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed that the gene network associated with the arginine metabolism pathway was enriched in livers infected by FAdV-4; 10 genes were downregulated and 8 genes were upregulated in these livers when compared to noninfected livers. The DEGs identified in livers were reanalyzed by real-time fluorescence quantitative PCR (qPCR); results indicated that the mRNA levels of the DEGs concurred with the data derived from KEGG analysis. Next, we used qPCR to detect the DEGs of the arginine metabolism pathway in a hepatocellular carcinoma cell line (LMH) after infection with FAdV-4 for 24 hr; this also indicated that the mRNA levels of the DEGs concurred with that seen in the liver. We also used si-RNA oligonucleotides to knock down the mRNA levels of iNOS in LMH cells infected with FAdV-4 and found that the viral load of FAdV-4 was increased. Further investigation revealed that the addition of 240 µg/ml of arginine into the culture medium of LMH cells infected with FAdV-4 for 24 hr led to a significant increase in the mRNA levels of iNOS but a significant reduction in the viral load of FAdV-4. Therefore, our data indicated that when broiler chickens become infected with FAdV-4, the arginine metabolic pathway in the liver becomes dysfunctional and the iNOS mRNA level decreases. This will add benefit to the replication of FAdV-4 but can be inhibited by the addition of an appropriate amount of arginine.

Synthesis of Monovalent N-Acetylgalactosamine Phosphoramidite for Liver-Targeting Oligonucleotides.

Ligand-targeted drug delivery (LTDD) has emerged as an attractive option in the field of oligonucleotide drugs following the great success of N-acetylgalactosamine (GalNAc)-conjugated siRNA and antisense oligonucleotides. GalNAc is a well-known ligand of the asialoglycoprotein receptor (ASGPR), and is classified as a C-type lectin associated with the metabolism of desialylated glycoproteins. This article describes the synthesis of a non-nucleosidic monovalent GalNAc phosphoramidite-a useful reagent for facilitating the conjugation of GalNAc epitopes into oligonucleotides using DNA synthesizers-together with some important caveats. The monomeric GalNAc consists of three parts: (1) a GalNAc moiety, (2) a linker moiety, and (3) a trans-4-hydroxyprolinol (tHP) branch point. The GalNAc moiety and the tHP moiety are coupled via a condensation reaction to prepare the monovalent GalNAc phosphoramidite. © 2019 by John Wiley & Sons, Inc. Basic Protocol 1: Synthesis of N-acetylgalactosamine ligand Basic Protocol 2: Preparation of trans-4-hydroxyprolinol building block Basic Protocol 3: Preparation of GalNAc phosphoramidite.

Orphan designation: Synthetic double-stranded siRNA oligonucleotide directed against p53 mRNA (teprasiran), Prevention of delayed graft function after renal transplantation

Orphan designation: Synthetic double-stranded siRNA oligonucleotide directed against p53 mRNA (teprasiran), Prevention of delayed graft function after renal transplantation

Kinome-Wide Screening with Small Interfering RNA Identified Polo-like Kinase 1 as a Key Regulator of Proliferation in Oral Cancer Cells.

Oral squamous cell carcinoma (OSCC) is one of the major leading causes of cancer-related death worldwide, with limited effective markers for diagnosis and therapy, which has caused a low overall survival rate in the past decades. Kinases play important roles in tumor development and malignancy in various types of cancer. However, little is known about the role of kinases in OSCC cells. In this study, an arrayed kinome small interfering RNA (siRNA) library was used to screen oral cancer cell lines and counter assayed with normal fibroblast cells to identify the genes required for cancer cell proliferation. We found that polo-like kinase 1 (PLK1) was one of the most potent genes required for OSCC cell proliferation. The knockdown of PLK1 with a siRNA or antisense oligonucleotide (ASO) consistently diminished cyclin-B1 (CCNB1) expression/phosphorylation and the G2-M phase transition. Similar effects were observed in cells treated with the PLK1 kinase inhibitor BI6727. Besides, The Cancer Genome Atlas (TCGA) analysis revealed that PLK1 was elevated in tumor tissues and associated with short survival in patients with OSCC. We also found that PLK1 expression was highly correlated with the expression of its downstream effector, CCNB1, in patients with OSCC. Coexpression of the two genes resulted in a poor prognosis of OSCC patients, particularly those in the advanced stages of OSCC. Taken together, our results suggest that PLK1 might be a diagnostic or therapeutic marker for OSCC.

Long Noncoding RNA, ANRIL, Regulates the Proliferation of Head and Neck Squamous Cell Carcinoma.

ANRIL is a long noncoding RNA located on INK4 locus, which encodes p15 and p16 that cause G1 phase arrest in the cell cycle. ANRIL positively regulates proliferation of several kinds of cancer cells such as lung and gastric cancers. This study, examined the effect of ANRIL in head and neck squamous cell carcinoma cells. Cells were transfected with siRNA oligonucleotides targeting ANRIL. Transfected cells were subjected to cell-cycle and quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analysis. Depletion of ANRIL increased p15 mRNA in FaDu cells, and p15 and p16 mRNA in CAL27 cells and inhibited proliferation of these cells. Cell cycle analysis showed that depletion of ANRIL caused arrest at the G1 phase of the cell cycle. ANRIL promotes G1 phase progression by repressing p15 and p16, and thus promotes FaDu and CAL27 cell proliferation.