Antisense Technology Research Articles

Depletion of the Long Non-Coding RNA MALAT1 primes Chronic Myelomonocytic Leukemia (CMML) for Differentiation Therapy with All-Trans retinoic Acid (ATRA) through the Transcription Factor CREB

Improvements in antisense technology have now enabled clinically relevant therapeutic credentialing of the noncoding genome. MALAT1 is a long non-coding RNA that, among other functions, is thought to serve as a nuclear scaffold for splicing and transcription factors. MALAT1 expression is associated with inferior prognosis across solid tumors and its depletion impairs proliferation and metastasis in preclinical solid tumor models. We found that elevated MALAT1 levels are independently associated with inferior overall survival in patients with CMML. Further, RNA-sequencing of primary CMML monocytes identified MALAT1 as the fourth most over-expressed transcript compared to controls. Therefore, we explored the biologic relevance and therapeutic candidacy of MALAT1 across several human and murine models of CMML. First, we crossed NRASQ61R/+Mx1-cre driven mice, which display a CMML-like phenotype, to MALAT1KO/KOmice. Although MALAT1KO/KOmice did not have abnormalities in complete blood counts, immunophenotyping of the hematopoietic stem cell (HSC) compartment identified statistically significantly lower numbers of HSC compared to wild type (WT) controls and a non-significant decrease in NRASQ61R/+/MALAT1KO/KOcompared to NRASQ61R/+alone. This decrease in HSC was not a result of impaired self-renewal as no differences were observed in these models after in vivo competitive transplant experiments. Therefore, we reasoned that MALAT1 expression may be controlling HSC differentiation. To test this, we transformed bone marrow cells from these models with an estrogen-regulated (ER) Hoxb8 construct enabling cells to maintain an HSC state until ER is withdrawn and myeloid differentiation is induced. ER-Hoxb8NRASQ61R/+/MALAT1KO/KOcells had increased basal levels of Gr-1 compared to ER-Hoxb8 transformed NRASQ61R/+alone that was dramatically enhanced upon ER withdrawal suggesting that MALAT1 depletion regulates myeloid differentiation. These findings were validated by assessment of morphology, transcriptome, and in vivo immunophenotyping of bone marrow and spleen cells. Further, moribund NRASQ61R/+/MALAT1KO/KOmice displayed a reduction in organomegaly typically associated with leukemic burden. We validated this in human monocytic leukemia by generating MALAT1 depleted THP-1 isogeneic cell lines using the CRISPR/Cas9 system. MALAT1 depleted THP-1 cells (MKO) had greater terminal differentiation according to immunophenotypic markers and morphology that was greatly enhanced when treated with phorbol myristate acetate. Last, MKO orthotopic xenografts demonstrated inferior human leukemia engraftment and decreased spleen and liver weights, and heterotopic xenografts exhibited reduced tumor volume, collectively suggesting diminished leukemic burden. Because ATRA has been clinically tested in CMML with minimal effects, we next explored whether MALAT1 depletion could potentiate ATRA differentiation in CMML. First, we treated MKO cells with ATRA and observed a large induction of myeloid differentiation by marker expression and morphologic assessment compared to isogenic controls. This was validated by NRASQ61R/+/MALAT1KO/KOmice demonstrating that ATRA more robustly induced differentiation compared to vehicle which was not seen in NRASQ61R/+/MALAT1+/+mice. Next, we tested MALAT1 antisense oligonucleotides (ASOs) currently under clinical development in THP-1 cells +/- ATRA and demonstrated both an increase in myeloid differentiation and apoptosis compared to ATRA alone. To test this therapeutic strategy in primary CMML specimens, we generated CMML patient-derived xenografts (n=30 mice) and treated each with ASO, ATRA, the combination, or controls and identified a more robust reduction in human HSC engraftment with the combination. To explore the mechanistic basis for these findings, we performed RNA-sequencing of MALAT1-depleted or control cells and identified that CREB target genes were differentially expressed. Basal protein levels of p-CREB were also decreased in MKO cells and were further reduced in the nucleus of MKO by western and microscopy. Lastly, overexpression of WT or constitutively active CREB but not its dominant negative rescued the differentiation effect seen in ATRA treated MKO cells. Taken together, MALAT1 is a novel, CREB-dependent regulator of myeloid differentiation and its depletion potentiates ATRA therapy. Disclosures Cluzeau: Menarini: Consultancy; Jazz Pharma: Consultancy; Abbvie: Consultancy. Komrokji:celgene: Consultancy; pfizer: Consultancy; DSI: Consultancy; JAZZ: Speakers Bureau; Novartis: Speakers Bureau; JAZZ: Consultancy; Agios: Consultancy; Incyte: Consultancy. MacLeod:Ionis Pharmaceuticals: Employment. List:Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding. Epling-Burnette:Forma Therapeutics: Research Funding; Celgene Corporation: Patents & Royalties, Research Funding; Incyte Corporation: Research Funding.

Rationale and Design of a Phase 3 Study to Evaluate the Efficacy and Safety of ION-682884 in Patients with Transthyretin-Mediated Amyloid Cardiomyopathy (ATTR-CM)

Background: Transthyretin amyloidosis cardiomyopathy (ATTR-CM) is a life-threatening, irreversible condition, which can lead to heart failure (HF) and, ultimately, heart transplant or death. Despite the recent approval in United States of a TTR stabilizer (VYNDAQEL®-tafamidis meglumine;VYNDAMAX™-tafamidis) for the treatment of ATTR-CM, disease progression still occurs. This study aims to determine if treatment with AKCEA-TTR-LRx (ION-682884), an antisense oligonucleotide (ASO), is safe and superior to placebo in reducing the risk of cardiovascular (CV) death or CV clinical events in patients with hereditary (hATTR-CM) or wild-type ATTR-CM (wtATTR-CM). Study Design and Methods: AKCEA-TTR-LRx (ION-682884) is a follow-on compound that incorporates the Ligand-Conjugated Antisense (LICA) technology; in this case, a triantennary N-acetyl galactosamine (GalNAc) moiety which targets the asialoglycoprotein receptors (ASGPR) expressed abundantly on the hepatocyte cell surface. In comparison to inotersen, its parent compound, ION-682884 requires a lower dose and frequency of administration (27-fold smaller; 45mg SC Q4W) to achieve a similar reduction in ATTR, providing greater patient convenience. ION-682884-CS2 (EudraCT No: 2019-002835-27) is a Phase 3 global, double-blind, randomized, placebo-controlled study assessing the efficacy and safety of AKCEA-TTR-LRx (ION-682884) in hATTR-CM or wtATTR-CM patients receiving available background standard of care (SoC) therapy. Approximately 750 patients with a history of HF due to ATTR-CM will be randomized 1:1 to receive AKCEA-TTR-LRx (ION-682884) or placebo administered by subcutaneous injection once every 4 weeks. The main inclusion criteria include confirmed diagnosis of ATTR-CM by tissue biopsy or positive PYP/DPD scan, end-diastolic interventricular septum thickness of >12mm, NT-proBNP >600 pg/mL, NYHA class I-III and 6-minute walk distance (6MWD) >150 m. The main exclusion criteria include estimated glomerular filtration rate (eGFR) < 30 mL/min/1.73m2, platelet count below the low limit of normality and urine protein/creatinine ratio (UPCR) ≥ 1000 mg/g. Patients are allowed to concomitantly receive tafamidis/tafamidis meglumine as SoC for ATTR-CM, if locally approved and available, per physician's discretion. The study consists of a 120-week Treatment Period and a 20-week Post-Treatment Evaluation Period. During each study visit, subjects will undergo laboratory tests, cardiac assessments (echocardiography), and functional evaluations. Patient-reported outcomes (PRO) will also be collected. Primary efficacy endpoint is the composite of CV mortality and frequency of CV clinical events (HF-related urgent visits requiring administration of IV diuretics and/or CV-related hospitalizations) at Week 120 study visit, analyzed by the Finkelstein-Shoenfeld method. This test is based on the principle of each patient in the study being compared with every other patient in a pairwise manner in hierarchical fashion. Secondary endpoints include the change from baseline in the 6MWD, Kansas City Cardiomyopathy Questionnaire score, rate of CV mortality, CV clinical events, and all-cause of mortality at Week 120. Additional exploratory endpoints include a change from baseline in cardiac imaging parameters, renal function, biomarkers, and PROs questionnaires and disease scores. An interim analysis on change from baseline in 6MWD is also planned at Week 60. All deaths and CV clinical events will be adjudicated by an independent, blinded Clinical Adjudication Committee, using predefined endpoint criteria. Conclusions: Despite recent advances, there is still a need for more efficacious, safe and convenient treatment options for ATTR-CM. The ION-682884-CS2 is a large Phase 3 trial designed to evaluate the clinical efficacy and safety of AKCEA-TTR-LRx (ION-682884) compared to placebo for the treatment of ATTR-CM. Figure Disclosures Falk: Ionis Pharmaceuticals: Consultancy. Gertz:Ionis/Akcea: Consultancy; Alnylam: Consultancy; Proclara: Membership on an entity's Board of Directors or advisory committees; Prothena Biosciences Inc: Consultancy; Celgene: Consultancy; Janssen: Consultancy; Spectrum: Consultancy, Research Funding; Annexon: Consultancy; Appellis: Consultancy; Amgen: Consultancy; Medscape: Consultancy, Speakers Bureau; Physicians Education Resource: Consultancy; Abbvie: Other: personal fees for Data Safety Monitoring board; Research to Practice: Consultancy; Teva: Speakers Bureau; Johnson and Johnson: Speakers Bureau; DAVA oncology: Speakers Bureau; Pharmacyclics: Membership on an entity's Board of Directors or advisory committees; i3Health: Other: Development of educational programs and materials; Springer Publishing: Patents & Royalties; Amyloidosis Foundation: Research Funding; International Waldenstrom Foundation: Research Funding. Benson:Ionis Pharmaceuticals: Research Funding. Buchele:Ionis Pharmaceuticals: Employment. Brambatti:Ionis Pharmaceuticals: Employment. Tsimikas:Ionis Pharmaceuticals: Employment. Viney:Ionis Pharmaceuticals: Employment. Tai:Ionis Pharmaceuticals: Employment. Monteiro:Ionis Pharmaceuticals: Employment. Yang:Ionis Pharmaceuticals: Employment. O'Dea:Akcea Therapeutics: Employment. Karwatowska-Prokopczuk:Akcea Therapeutics: Employment. Schneider:Ionis Pharmaceuticals: Employment. Geary:Ionis Pharmaceuticals: Employment. Monia:Ionis Pharmaceuticals: Employment.

AdeB efflux pump gene knockdown by mRNA mediated peptide nucleic acid in multidrug resistance Acinetobacter baumannii

Multidrug-resistant Acinetobacter baumannii isolates cause critical problems in health-care environments. AdeABC is a resistance-nodulation-cell division (RND)-type multidrug efflux pump conferring resistance to clinically essential antibiotics in A. baumannii, such as ciprofloxacin. This study aimed to target adeB gene with antisense peptide nucleic acid (PNA) and investigate its effect on resistance to antibiotics. NCBI database was used to design appropriate PNA to target adeB gene, by connecting PNA to mRNA, the translation of mRNA can be prevented. Three clinical isolates and A. baumannii ATCC 17978 were treated with the designed PNA by electroporation and competence procedure. Minimum Inhibitory concentration (MIC) of ciprofloxacin, colistin, and tetracycline were determined by microbroth dilution method. In addition, the expression level of adeB gene was measured by quantitative real-time PCR (qRT-PCR). Isolates used in this study had mutations in gyrA and parC genes corresponding to resistance to ciprofloxacin. MIC of resistance to ciprofloxacin after treatment with PNA was reduced from 32 μg/ml to16 μg/ml in A. baumannii ATCC 17978 isolate. Susceptibility level of tetracycline, in the 2 clinical isolates was decreased from 64 μg/ml to 32 μg/ml and in the other isolate was reduced from 128 μg/ml to 64 μg/ml. The expression level of adeB gene was decreased in A. baumannii ATCC 17978 (P > 0.01) but not in clinical isolate (P = 0.107). Findings of the present study indicate overexpression of adeB efflux pump has extra effect on resistance to antibiotics in isolates with a defined mechanism of resistance. Antisense technology is a feasible technique to suppress the function of these genes, which may be further exploited to control multidrug-resistant isolates.

Development of gapmer antisense oligonucleotide with deoxyribonucleic guanidine (DNG) modifications

The properties of gapmer antisense oligonucleotide (ASO) flanked by deoxyribonucleic guanidine (DNG) were investigated for the potential application in antisense technology. DNG is a unique nucleotide analog which has a positively charged internucleotide guanidinium linkage instead of negatively charged phosphodiester backbone linkage. We prepared a gapmer ASO containing DNG units at both wings of the sequence and compared its properties with 2′,4′-BNA/LNA gapmer ASOs with phosphorothioate (PS) backbone. Although DNG gapmer showed no stabilizing effect on the duplex formation with target RNA, the DNG modification was found to be tolerant to exonuclease digestion. Furthermore, DNG gapmer can induce RNase H-mediated cleavage of target RNA molecule, a requisite property for the antisense strategy. Therefore, the DNG gapmer developed in this study could be an interesting and useful candidate for the development of potent ASOs.

Therapeutic strategies for targeting telomerase in cancer.

Telomere and telomerase play important roles in abnormal cell proliferation, metastasis, stem cell maintenance, and immortalization in various cancers. Therefore, designing of drugs targeting telomerase and telomere is of great significance. Over the past two decades, considerable knowledge regarding telomere and telomerase has been accumulated, which provides theoretical support for the design of therapeutic strategies such as telomere elongation. Therefore, the development of telomere-based therapies such as nucleoside analogs, non-nucleoside small molecules, antisense technology, ribozymes, and dominant negative human telomerase reverse transcriptase are being prioritized for eradicating a majority of tumors. While the benefits of telomere-based therapies are obvious, there is a need to address the limitations of various therapeutic strategies to improve the possibility of clinical applications. In this study, current knowledge of telomere and telomerase is discussed, and therapeutic strategies based on recent research are reviewed.

Assessment of the functional impact on the pre-mRNA splicing process of 28 nucleotide variants associated with Pompe disease in GAA exon 2 and their recovery using antisense technology.

Glycogen storage disease II (GSDII), also called Pompe disease, is an autosomal recessive inherited disease caused by a defect in glycogen metabolism due to the deficiency of the enzyme acid alpha-glucosidase (GAA) responsible for its degradation. So far, more than 500 sequence variants of the GAA gene have been reported but their possible involvement on the pre-messenger RNA splicing mechanism has not been extensively studied. In this work, we have investigated, by an in vitro functional assay, all putative splicing variants within GAA exon 2 and flanking introns. Our results show that many variants falling in the canonical splice site or the exon can induce GAA exon 2 skipping. In these cases, therefore, therapeutic strategies aimed at restoring protein folding of partially active mutated GAA proteins might not be sufficient. Regarding this issue, we have tested the effect of antisense oligonucleotides (AMOs) that were previously shown capable of rescuing splicing misregulation caused by the common c.-32-13T>G variant associated with the childhood/adult phenotype of GSDII. Interestingly, our results show that these AMOs are also quite effective in rescuing the splicing impairment of several exonic splicing variants, thus widening the potential use of these effectors for GSDII treatment.

The Application of Microfluidics in Preparing Nano Drug Delivery Systems

The Application of Microfluidics in Preparing Nano Drug Delivery Systems

MRNA levels can be reduced by antisense oligonucleotides via no-go decay pathway.

Antisense technology can reduce gene expression via the RNase H1 or RISC pathways and can increase gene expression through modulation of splicing or translation. Here, we demonstrate that antisense oligonucleotides (ASOs) can reduce mRNA levels by acting through the no-go decay pathway. Phosphorothioate ASOs fully modified with 2′-O-methoxyethyl decreased mRNA levels when targeted to coding regions of mRNAs in a translation-dependent, RNase H1-independent manner. The ASOs that activated this decay pathway hybridized near the 3′ end of the coding regions. Although some ASOs induced nonsense-mediated decay, others reduced mRNA levels through the no-go decay pathway, since depletion of PELO/HBS1L, proteins required for no-go decay pathway activity, decreased the activities of these ASOs. ASO length and chemical modification influenced the efficacy of these reagents. This non-gapmer ASO-induced mRNA reduction was observed for different transcripts and in different cell lines. Thus, our study identifies a new mechanism by which mRNAs can be degraded using ASOs, adding a new antisense approach to modulation of gene expression. It also helps explain why some fully modified ASOs cause RNA target to be reduced despite being unable to serve as substrates for RNase H1.

Two tales of Annexin A2 knock-down: One of compensatory effects by antisense RNA and another of a highly active hairpin ribozyme

Besides altering its own expression during cell transformation, Annexin A2 is upregulated during the progression of many cancer types and also plays key roles during viral infection and multiplication. Consequently, there has been great interest in Annexin A2 as a potential drug target. The successful design of efficient in vivo delivery systems constitutes an obstacle in full exploitation of antisense and RNA-cleaving technologies for the knock-down of specific targets. Efficiency is dependent on the method of delivery and accessibility of the target. Here, hairpin ribozymes and an antisense RNA against rat annexin A2 mRNA were tested for their efficiencies in a T7-driven coupled transcription/translation system. The most efficient ribozyme and antisense RNA were subsequently inserted into a retroviral vector under the control of a tRNA promoter, in a cassette inserted between retroviral Long Terminal Repeats for stable insertion into host DNA. The Phoenix package system based on defective retroviruses was used for virus-mediated gene transfer into PC12 cells. Cells infected with the ribozyme-containing particles died shortly after infection. However, the same ribozyme showed a very high catalytic effect in vitro in cell lysates, explained by its loose hinge helix 2 region. This principle can be transferred to other ribozymes, such as those designed to cleave the guide RNA in the CRISPR/Cas9 technology, as well as to target specific viral RNAs. Interestingly, efficient down-regulation of the expression of Annexin A2 by the antisense RNA resulted in up-regulation of Annexin A7 as a compensatory effect after several cell passages. Indeed, compensatory effects have previously been observed during gene knock-out, but not during knock-down of protein expression. This highlights the problems in interpreting the phenotypic effects of knocking down the expression of a protein. In addition, these data are highly relevant when considering the effects of the CRISPR/Cas9 approach.

AcrAB–TolC Inhibition by Peptide-Conjugated Phosphorodiamidate Morpholino Oligomers Restores Antibiotic Activity in Vitro and in Vivo

Overexpression of bacterial efflux pumps is a driver of increasing antibiotic resistance in Gram-negative pathogens. The AcrAB-TolC efflux pump has been implicated in resistance to a number of important antibiotic classes including fluoroquinolones, macrolides, and β-lactams. Antisense technology, such as peptide-conjugated phosphorodiamidate morpholino oligomers (PPMOs), can be utilized to inhibit expression of efflux pumps and restore susceptibility to antibiotics. Targeting of the AcrAB-TolC components with PPMOs revealed a sequence for acrA, which was the most effective at reducing antibiotic efflux. This acrA-PPMO enhances the antimicrobial effects of the levofloxacin and azithromycin in a panel of clinical Enterobacteriaceae strains. Additionally, acrA-PPMO enhanced azithromycin in vivo in a K.pneumoniae septicemia model. PPMOs targeting the homologous resistance-nodulation-division (RND)-efflux system in P.aeruginosa, MexAB-OprM, also enhanced potency to several classes of antibiotics in a panel of strains and in a cell culture infection model. These data suggest that PPMOs can be used as an adjuvant in antibiotic therapy to increase the efficacy or extend the spectrum of useful antibiotics against a variety of Gram-negative infections.

Targeting Tumor Markers with Antisense Peptides: An Example of Human Prostate Specific Antigen

The purpose of this paper was to outline the development of short peptide targeting of the human prostate specific antigen (hPSA), and to evaluate its effectiveness in staining PSA in human prostate cancer tissue. The targeting of the hPSA antigen by means of antisense peptide AVRDKVG was designed according to a three-step method involving: 1. The selection of the molecular target (hPSA epitope), 2. the modeling of an antisense peptide (paratope) based on the epitope sequence, and 3. the spectroscopic evaluation of sense–antisense peptide binding. We then modified standard hPSA immunohistochemical staining practice by using a biotinylated antisense peptide instead of the standard monoclonal antibody and compared the results of both procedures. Immunochemical testing on human tissue showed the applicability of the antisense peptide technology to human molecular targets. This methodology represents a new approach to deriving peptide ligands and potential lead compounds for the development of novel diagnostic substances, biopharmaceuticals and vaccines.

Transient Down-Regulation of Nucleoside Transporter 3 Gene Expression as a Drug Target in Leishmania major Using Antisense RNA Technology

This study was aimed to silencing the Nucleoside transporter 3 (NT3) permease nucleobases involved in the salvage pathway of Leishmania in order to disrupt purine nucleotide uptake in the parasite and consequently, destruction of the parasite. Overall, 502 bp fragment of the NT3 gene sequence was designed to produce an antisense transcript upon entry of the vector into the parasite. The NT3 construct was transfected into L. major promastigotes and NT3 gene expression was studied in vivo and in vitro conditions. Relative expression NT3 gene in transgenic Leishmania was decreased in tenth day. The percentages and the number of amastigotes infected macrophages with transgenic L. major were less than infected macrophages with wild-type strain. Our results in two groups of BALB/c female mice (wild-type strain and mutant, n=4 each group) were showed that size and number of ulcers in BALB/c mice infected with transgenic Leishmania promastigotes were less than the BALB/c mice infected with wild-type parasites. The results indicate the use of antisense RNA reduces of NT3 gene expression in L. major. More studies are required to obtain a new approach for treating Leishmania infection.

Clinical Significance of BRAF and ZEB2 Expressions in Healthy Adjacent Tissue of Bladder Cancer

Background: Numerous molecular changes are involved in the development andprogression of bladder cancer. Regular follow-up of patients is crucial due to the highrecurrence rate of bladder cancer. The aim of this study is to determine the role ofB-Raf proto-oncogene, serine/threonine kinase and ZEB2 expressions in onset andprogression of bladder cancer. We have also investigated their relationships topathological characteristics.Methods: We conducted this case-control study on bladder cancer and its healthyadjacent tissue, and normal bladder tissue from patients with benign prostatic hyperplasia.After extraction of total RNA and cDNA synthesis, quantitative expression analysis wasperformed in duplicate using real-time PCR. Changes in the gene expression werecalculated according to the 2(-ΔΔCt) equation. The products were confirmed by 1% agarosegel electrophoresis and sequenced by Bioneer Company. Data was analyzed using theSPSS software (version 16).Results: There was significantly greater B-Raf proto-oncogene, serine/threoninekinase expression in 82% of bladder tumor samples compared to the adjacent tissues.In 91.1% of tumor samples, the gene expression was also significantly higher than healthybladder tissues from patients with benign prostatic hyperplasia. We observedoverexpression of B-Raf proto-oncogene, serine/threonine kinase in 61.7% of thehealthy margin tissue samples compared to healthy bladder tissues of patients with benignprostatic hyperplasia (P<0.001). Expression of ZEB2 in 52.9% of the bladder tumorsamples was significantly higher than healthy peripheral tissues. This increase wasobserved in 94.1% of tumor samples compared to healthy bladder tissues of patientswith benign prostatic hyperplasia (P<0.001). Pearson correlation coefficient showeda positive relationship between B-Raf proto-oncogene, serine/threonine kinase and ZEB2in cancerous samples (r = 0.75) and healthy margin tissue samples (r = 0.49).Conclusion: During the carcinogenesis process, molecular changes are seen inhealthy margin tissue. These molecular changes may be the reason for the highrecurrence rate of bladder cancer. B-Raf proto-oncogene, serine/threonine kinase canpotentially be a target cancer therapy in antisense technologies.

Tandem Requirement for Renal D1R and D5R Functionality

The peripheral dopaminergic system assists in maintaining blood pressure homeostasis by engendering natriuresis mainly through the D1R and D5R receptors. These receptors share structural features and pharmacological profiles; there is no specific agonist that can discriminate between these two receptors. Genetic ablation of one receptor in the kidney results in the loss of activity of the other. Therefore, we evaluated the mechanisms for the cooperative and co‐dependent interaction between these two receptors. Both receptors share the same anatomical distribution, specifically at the brush border and luminal membranes of renal proximal tubules and collecting ducts, respectively. These receptors dimerized (~110 kDa) and coimmunoprecipitated each other, basally and after fenoldopam (D1R/D5R agonist, 1 μM/15 min) treatment. Bimolecular fluorescence complementation showed that heterologously expressed D1R and D5R tagged with the C and N termini of the fluorescent protein EYFP, respectively, heterodimerized basally in HEK‐293 cells. This was corroborated by confocal microscopy of HEK‐293 cells that were transiently transfected with D1R::RFP and D5R::GFP, which showed colocalization basally at the plasma membrane, most abundantly at the apical membrane in polarized cells. The D1R and D5R partitioned to lipid and non‐lipid rafts; agonist treatment enhanced the lipid raft segregation of D1R and D5R. Both receptors were abundantly expressed in renal proximal tubules of human and C57Bl/6 mouse kidneys, where they colocalized basally that was increased by fenoldopam (1 μM/15 min). D1R/D5R stimulation is known to increase cAMP production and inhibit renal sodium transport. Receptor gene silencing via antisense technology in human renal proximal tubule cells (hRPTCs) decreased the cAMP response to fenoldopam (−71±2% for D1R, vs. −38±7% for D5R, vs. −90±2% for both, *P&lt;0.05). In hRPTCs grown in Transwells, fenoldopam or ouabain (500 nM/30 min) given at the basolateral compartment inhibited Na+/K+‐ATPase (NKA) activity and increased intracellular Na+. siRNA‐mediated gene silencing of DRD1/DRD5 prevented the inhibition of NKA activity by fenoldopam (*P&lt;0.05). Our observations demonstrate the requirement for D1R/D5R to form heterodimers to achieve their full functionality.Support or Funding InformationR01DK039308, R01HL092196, and P01HL074940This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Antisense Oligonucleotide: Basic Concepts and Therapeutic Application in Inflammatory Bowel Disease.

Several molecular technologies aimed at regulating gene expression that have been recently developed as a strategy to combat inflammatory and neoplastic diseases. Among these, antisense technology is a specific, rapid, and potentially high-throughput approach for inhibiting gene expression through recognition of cellular RNAs. Advances in the understanding of the molecular mechanisms that drive tissue damage in different inflammatory diseases, including Crohn’s disease (CD) and ulcerative colitis (UC), the two major inflammatory bowel diseases (IBDs) in humans, have facilitated the identification of novel druggable targets and offered interesting therapeutic perspectives for the treatment of patients. This short review provides a comprehensive understanding of the basic concepts underlying the mechanism of action of the oligonucleotide therapeutics, and summarizes the available pre-clinical and clinical data for oligonucleotide-based therapy in IBD.

Long non-coding RNAs are emerging targets of phytochemicals for cancer and other chronic diseases.

The long non-coding RNAs (lncRNAs) are the crucial regulators of human chronic diseases. Therefore, approaches such as antisense oligonucleotides, RNAi technology, and small molecule inhibitors have been used for the therapeutic targeting of lncRNAs. During the last decade, phytochemicals and nutraceuticals have been explored for their potential against lncRNAs. The common lncRNAs known to be modulated by phytochemicals include ROR, PVT1, HOTAIR, MALAT1, H19, MEG3, PCAT29, PANDAR, NEAT1, and GAS5. The phytochemicals such as curcumin, resveratrol, sulforaphane, berberine, EGCG, and gambogic acid have been examined against lncRNAs. In some cases, formulation of phytochemicals has also been used. The disease models where phytochemicals have been demonstrated to modulate lncRNAs expression include cancer, rheumatoid arthritis, osteoarthritis, and nonalcoholic fatty liver disease. The regulation of lncRNAs by phytochemicals can affect multi-steps of tumor development. When administered in combination with the conventional drugs, phytochemicals can also produce synergistic effects on lncRNAs leading to the sensitization of cancer cells. Phytochemicals target lncRNAs either directly or indirectly by affecting a wide variety of upstream molecules. However, the potential of phytochemicals against lncRNAs has been demonstrated mostly by preclinical studies in cancer models. How the modulation of lncRNAs by phytochemicals produce therapeutic effects on cancer and other chronic diseases is discussed in this review.

Structural insight into antisense gapmer-RNA oligomer duplexes through molecular dynamics simulations

There is an extensive research carrying out on antisense technology and the molecules entering into clinical trials are increasing rapidly. Phosphorothioate (PS) is a chemical modification in which nonbridged oxygen is replaced with a sulfur, consequently providing resistance against nuclease activity. The 2'-4' conformationally restricted nucleoside has the structural features of both 2'-O-methoxy ethyl RNA (MOE), which shows good toxicity profile, and locked nucleic acid (LNA), which shows good binding affinity towards the target RNA. These modifications have been studied and suggested that they can be a potential therapeutic agents in antisense therapy. Mipomersen (ISIS 301012), which contains the novel nucleoside modification has been used to target to apolipoprotein (Apo B), which reduces LDL cholesterol by 6–41%. In this study, classical molecular dynamics (MD) simulations were performed on six different antisense gapmer/target-RNA oligomer duplexes (LNA-PS-LNA/RNA, RcMOE-PS-RcMOE/RNA, ScMOE-PS-ScMOE/RNA, MOE-PS-MOE/RNA, PS-DNA/RNA and DNA/RNA) to investigate the structural dynamics, stability and solvation properties. The LNA, MOE nucleotides present in respective duplexes are showing the structure of A-form and the PS-DNA nucleotides resemble the structure of B-form helix with respect to some of the helical parameters. Free energy calculations suggest that the oligomer, which contains LNA binds to the RNA strongly than other modifications as shown in experimental results. The MOE modified nucleotide, which although had a lower binding affinity but higher solvent accessible surface area (SASA) compared to the other modifications, may be influencing the toxicity and hence may be used it in Mipomersen, the second antisense molecule which is approved by FDA.Communicated by Ramaswamy H. Sarma

Blood-derived plasminogen drives brain inflammation and plaque deposition in a mouse model of Alzheimer’s disease

Two of the most predominant features of the Alzheimer's disease (AD) brain are deposition of β-amyloid (Aβ) plaques and inflammation. The mechanism behind these pathologies remains unknown, but there is evidence to suggest that inflammation may predate the deposition of Aβ. Furthermore, immune activation is increasingly being recognized as a major contributor to the pathogenesis of the disease, and disorders involving systemic inflammation, such as infection, aging, obesity, atherosclerosis, diabetes, and depression are risk factors for the development of AD. Plasminogen (PLG) is primarily a blood protein synthesized in the liver, which when cleaved into its active form, plasmin (PL), plays roles in fibrinolysis, wound healing, cell signaling, and inflammatory regulation. Here we show that PL in the blood is a regulator of brain inflammatory action and AD pathology. Depletion of PLG in the plasma of an AD mouse model through antisense oligonucleotide technology dramatically improved AD pathology and decreased glial cell activation in the brain, whereas an increase in PL activity through α-2-antiplasmin (A2AP) antisense oligonucleotide treatment exacerbated the brain's immune response and plaque deposition. These studies suggest a crucial role for peripheral PL in mediating neuroimmune cell activation and AD progression and could provide a link to systemic inflammatory risk factors that are known to be associated with AD development.

Correction of a Splicing Mutation Affecting an Unverricht-Lundborg Disease Patient by Antisense Therapy.

Unverricht-Lundborg disease (ULD) is a common form of progressive myoclonic epilepsy caused by mutations in the cystatin B gene (CSTB) that encodes an inhibitor of several lysosomal cathepsins. Presently, only pharmacological treatment and psychosocial support are available for ULD patients. To overcome the pathogenic effect of the ULD splicing mutation c.66G>A (exon 1), we investigated whether an antisense oligonucleotide therapeutic strategy could correct the defect in patient cells. A specific locked nucleic acid (LNA) antisense oligonucleotide was designed to block a cryptic 5′ss in intron 1. Overall, this approach allowed the restoration of the normal splicing pattern. Furthermore, the recovery was both sequence and dose-specific. In general, this work provides a proof of principle on the correction of a CSTB gene defect causing ULD through a mutation-specific antisense therapy. It adds evidence to the feasibility of this approach, joining the many studies that are paving the way for translating antisense technology into the clinical practice. The insights detailed herein make mutation-based therapy a clear candidate for personalized treatment of ULD patients, encouraging similar investigations into other genetic diseases.

The zinc-finger transcription factor BcMF20 and its orthologs in Cruciferae which are required for pollen development

Brassica campestris Male Fertility 20 (BcMF20) is a typical zinc-finger transcription factor that was previously isolated from flower buds of Chinese cabbage (Brassica campestris ssp. chinensis). By applying expression pattern analysis, it can be known that BcMF20 was specifically and strongly expressed in tapetum and pollen, beginning from the uninucleate stage, and was maintained during the mature-pollen stage. As BcMF20 was highly conserved in Cruciferae, it can be indicated that this zinc-finger transcription factor is important during the growth of Cruciferae. In this study, 12 C2H2-type zinc-finger TFs which shared high homology with BcMF20 were found from NCBI via BLAST. A new molecular phylogenetic tree was constructed by the comparison between BcMF20 and these 12 C2H2-type zinc-finger TFs with NJ method. By analyzing this phylogenetic tree, the evolution of BcMF20 was discussed. Then, antisense RNA technology was applied in the transgenesis of Arabidopsis thaliana to get the deletion mutants of BcMF20, so that its function during the pollen development can be identified. The results showed: BcMF20 are in the same clade with three genes from Arabidopsis. The inhibition of BcMF20 expression led to smaller amounts of and lower rate in germination of pollen and lower rate in fruit setting in certain transgenetic plants. This also led to the complete collapse of pollen grains. By SEM and TEM, pollen morphology and anther development processes were observed. In the middle uninucleate microspore stage, a relatively thin or even no primexine was formed in microspores. This may result in the malformation of the pollen wall and finally cause the deformity of pollens. Above all, it can be indicated that BcMF20 may act as a part of regulation mechanisms of TAZ1 and MS1. Together they play a role in a genetic pathway in the tapetum to act on proliferation of tapetal cells and keep the normal development of pollens.