Administration Of Antisense Oligonucleotides Research Articles

Abstract 3029: Targeting Annexin A4 with antisense oligonucleotides improves platinum resistance of ovarian cancer

Abstract [Introduction] Ovarian cancer (OvCa) is one of the main causes of gynecologic cancer death in many countries and the number of patients is increasing. The standard treatment for advanced OvCa is cytoreductive surgery and adjuvant chemotherapy. The key drug for OvCa chemotherapy is platinum. However more than 70% of advanced stage OvCa relapse within 5 years, and the major issue to treat them is platinum resistance. We previously reported that Annexin (Anx) A4 protein was associating with chemoresistance to platinum-based cancer drugs. Nucleic acid therapeutics is promising therapy to target molecule existing intra cell membrane. The aim of this study is to evaluate the usefulness of antisense oligonucleotides (ASOs) and toovercome the platinum chemoresistance with ASOs by suppressing the expression of Anx A4 in cancer cells. [Methods] Cell line: To assess AnxA4 ASOs, two human ovarian cancer cell lines, RMG-1 and OVISE, which have platinum resistance and strongly express Anx A4, were used in each experiment. Anx A4 ASOs: We selected targeting Annexin A4 mRNA sequence in silico and made 16 ASOs. To enhance the stability, 2’, 4’-bridged nucleic acid were inserted in both 3’and 5’ends. And we analyzed suppression of Annexin A4 in ovarian clear cell cancer cell lines in vitro using real time PCR and western blotting. Platinum resistance verification: In vitro, cells were transfected with ASOs using lipofectamine 2000 and were exposed to various concentrations of cisplatin (0 - 100 μM) for 72 hr. Then, drug concentrations resulting in a 50% inhibition of cell growth (IC50values) were calculated. In vivo, we used ICRnu/nu mice xenografted subcutaneously with RMG-I and OVISE cells. Intraperitoneal injection of cisplatin 3mg/kg after intratumoral administration of ASO 1mg/kg each twice a week were given to xenograft mice. [Result] Of the 16 types of antisense oligonucleotides, we focused on 2 type of ASO, #7, #9 which had stronger knockdown effect for Anx A4 expression. In RMG-I cell, IC50value of Anx A4 ASO transfected cells were 6.42 µM in compared with 11.12 µM in control ASO transfected cells. (p=0.012) In OVISE cells, IC50value was also significantly higher in transfected Anx A4 ASO cells. (10.30 µM, 4.66 µM, p<0.0001) Significantly more amount of platinum had accumulated in Anx A4 ASO transfected cells in compared with control ASO transfected cells and no treatment cells.In vivo, tumor growth of mice treated with cisplatin+Anx A4 ASOs were significantly inhibited compared to cisplatin + control ASOs in both the RMG-1 and OVISE cell lines xenograft mice. [Conclusion] By transfection of ANXA4 ASO, platinum resistance has been improved both in vitro and in vivo. Targeting ANX A4 with nucleic therapeutics could be an option for platinum resistant ovarian cancer. Citation Format: Satoshi Nakagawa, Reisa Kakubari, Shinya Matsuzaki, Yutaka Ueda, Kosuke Hiramatsu, Satoshi Serada, Minoru Fujimoto, Tadashi Kimura, Tetsuji Naka. Targeting Annexin A4 with antisense oligonucleotides improves platinum resistance of ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3029.

Hypothalamic nesfatin-1 mediates feeding behavior via MC3/4R-ERK signaling pathway after weight loss in obese Sprague-Dawley rats

Nesfatin-1 is an anorexic peptide derived from nucleobindin 2 (NUCB2). An increase in hypothalamic nesfatin-1 inhibits feeding behavior and promotes weight loss. However, the effects of weight loss on hypothalamic nesfatin-1 levels are unclear. In this study, obese rats lost weight in three ways: Calorie Restriction diet (CRD), Sleeve gastrectomy (SG) and Roux-en-Y gastric bypass (RYGB). We found an increase in nesfatin-1 serum and cerebrospinal fluid levels after weight loss in obese Sprague-Dawley (SD) rats. Moreover, weight loss also increased hypothalamic melanocortin 3/4 receptor (MC3/4R) and extracellular regulated kinase phosphorylation (p-ERK) signaling. Third ventricle administration of antisense morpholino oligonucleotide (MON) against the gene encoding NUCB2 inhibited hypothalamic nesfatin-1 and p-ERK signaling, increased food intake and reduced body weight loss in SG and RYGB obese rats. Third ventricle administration of SHU9119 (MC3/4R blocker) blocked hypothalamic MC3/4R, inhibited p-ERK signaling, increased food intake and reduced body weight loss in SG and RYGB obese rats. These findings indicate that weight loss leads to an increase in hypothalamic nesfatin-1. The increase in hypothalamic nesfatin-1 participates in regulating feeding behavior through the MC3/4R-ERK signaling especially after SG and RYGB.

S148 TFR2-HAPLOINSUFFICIENCY ENHANCES THE BENEFICIAL EFFECT OF TMPRSS6-ANTISENSE OLIGONUCLEOTIDE TREATMENT IN BETA-THALASSEMIA MICE

Background: Transferrin receptor 2 (TFR2) is a type II transmembrane protein mainly expressed in the liver and in the erythroid compartment. In the liver it acts as an activator of the iron hormone hepcidin and, when mutated, is responsible for type 3 hereditary hemochromatosis. In erythroid cells TFR2 binds the erythropoietin (EPO) receptor and serves as a brake of the EPO-EPOR signaling pathway. We have recently demonstrated that the complete loss of bone marrow (BM) Tfr2 increases erythroblast EPO sensitivity and improves anemia, ineffective erythropoiesis and iron-overload in a β-thalassemia intermedia murine model (Hbbth3/+), without reducing splenomegaly. On the contrary, approaches based on the induction of iron restriction were proven to be effective in ameliorating splenomegaly and anemia in thalassemic mice. Among these, targeting the hepcidin inhibitor TMPRSS6 is one of the most promising strategies. Aims: The main aim of the present work is to investigate whether the combination of Tfr2-haploinsufficiency in the BM and TMPRSS6 antisense oligonucleotide (ASOs) administration will reduce splenomegaly and provide a synergistic benefit on thalassemic erythropoiesis and anemia. Methods: We generated thalassemic mice with both or a single Tfr2 allele in the BM by transplanting BM cells from Hbbth3/+ or Tfr2+/-/Hbbth3/+ mutants into lethally irradiated wild-type mice. Mice were treated with TMPRSS6-ASO (or control ASO) twice a week for 6 weeks starting 9 weeks after BM transplantation. Complete blood count was determined at the beginning of the treatment and after 3 and 6 weeks. At sacrifice, blood and organs were collected for full phenotypic analysis. Results: Deletion of a single Tfr2 allele in Hbbth3/+ mice BM mildly increased Red Blood Cells (RBC) count and Hemoglobin (Hb) levels and improved erythropoiesis, with a concomitant decrease in EPO levels, but no reduction of spleen size. A similar phenotype was induced by TMPRSS6-ASO treatment of Hbbth3/+ animals, with additional partial correction of splenomegaly. The combination of BM Tfr2 haploinsufficiency and TMPRSS6-ASO had a striking effect on the thalassemic phenotype: indeed, an impressive increase in RBC count (up to 11.2 ± 1.2∗106/μl) and Hb (∼3 g/L) compared with Hbbth3/+ was attained, accompanied by the normalization of reticulocyte and EPO levels, improved erythropoiesis and spleen size reduction. Mean corpuscular volume and hemoglobin were strongly reduced. This indicates a disproportionate erythropoietic expansion relative to the available iron, whose circulating levels are decreased relative to untreated thalassemic controls. Summary/Conclusion: While Tfr2 haploinsufficiency does not impair systemic iron homeostasis, here we demonstrate that deletion of a single Tfr2 allele in the BM is sufficient to improve the hematological parameters of thalassemic mice. This finding paves the way toward a potential targeted therapy based on partial Tfr2 inhibition that might be beneficial for thalassemia (and potentially for other forms of anemia) without inducing defective hepcidin activation and iron overload. In addition, our results prove that targeting Tfr2 in combination with an anti-TMPRSS6 therapy may be a valuable therapeutic approach that strongly improves the thalassemic phenotype. However, Tmprss6 inhibition must be tightly controlled to avoid excessive iron restriction. Further experiments will allow setting up the optimal conditions.

Inhibition of the Hypoxia-Inducible Factor 1α-Induced Cardiospecific HERNA1 Enhance-Templated RNA Protects From Heart Disease.

Enhancers are genomic regulatory elements conferring spatiotemporal and signal-dependent control of gene expression. Recent evidence suggests that enhancers can generate noncoding enhancer RNAs, but their (patho)biological functions remain largely elusive. We performed chromatin immunoprecipitation-coupled sequencing of histone marks combined with RNA sequencing of left ventricular biopsies from experimental and genetic mouse models of human cardiac hypertrophy to identify transcripts revealing enhancer localization, conservation with the human genome, and hypoxia-inducible factor 1α dependence. The most promising candidate, hypoxia-inducible enhancer RNA ( HERNA)1, was further examined by investigating its capacity to modulate neighboring coding gene expression by binding to their gene promoters by using chromatin isolation by RNA purification and λN-BoxB tethering-based reporter assays. The role of HERNA1 and its neighboring genes for pathological stress-induced growth and contractile dysfunction, and the therapeutic potential of HERNA1 inhibition was studied in gapmer-mediated loss-of-function studies in vitro using human induced pluripotent stem cell-derived cardiomyocytes and various in vivo models of human pathological cardiac hypertrophy. HERNA1 is robustly induced on pathological stress. Production of HERNA1 is initiated by direct hypoxia-inducible factor 1α binding to a hypoxia-response element in the histoneH3-lysine27acetylation marks-enriched promoter of the enhancer and confers hypoxia responsiveness to nearby genes including synaptotagmin XVII, a member of the family of membrane-trafficking and Ca2+-sensing proteins and SMG1, encoding a phosphatidylinositol 3-kinase-related kinase. Consequently, a substrate of SMG1, ATP-dependent RNA helicase upframeshift 1, is hyperphoshorylated in a HERNA1- and SMG1-dependent manner. In vitro and in vivo inactivation of SMG1 and SYT17 revealed overlapping and distinct roles in modulating cardiac hypertrophy. Finally, in vivo administration of antisense oligonucleotides targeting HERNA1 protected mice from stress-induced pathological hypertrophy. The inhibition of HERNA1 postdisease development reversed left ventricular growth and dysfunction, resulting in increased overall survival. HERNA1 is a novel heart-specific noncoding RNA with key regulatory functions in modulating the growth, metabolic, and contractile gene program in disease, and reveals a molecular target amenable to therapeutic exploitation.

Heparin-binding EGF-like growth factor (HB-EGF) antisense oligonucleotide protected against hyperlipidemia-associated atherosclerosis

Background and aimsHeparin-binding EGF-like growth factor (HB-EGF) is a representative EGF family member that interacts with EGFR under diverse stress environment. Previously, we reported that the HB-EGF-targeting using antisense oligonucleotide (ASO) effectively suppressed an aortic aneurysm in the vessel wall and circulatory lipid levels. In this study, we further examined the effects of the HB-EGF ASO administration on the development of hyperlipidemia-associated atherosclerosis using an atherogenic mouse model. Methods and resultsThe male and female LDLR deficient mice under Western diet containing 21% fat and 0.2% cholesterol content were cotreated with control and HB-EGF ASOs for 12 weeks. We observed that the HB-EGF ASO administration effectively downregulated circulatory VLDL- and LDL-associated lipid levels in circulation; concordantly, the HB-EGF targeting effectively suppressed the development of atherosclerosis in the aorta. An EGFR blocker BIBX1382 administration suppressed the hepatic TG secretion rate, suggesting a positive role of the HB-EGF signaling for the hepatic VLDL production. We newly observed that there was a significant improvement of the insulin sensitivity by the HB-EGF ASO administration in a mouse model under the Western diet as demonstrated by the improvement of the glucose and insulin tolerances. ConclusionThe HB-EGF ASO administration effectively downregulated circulatory lipid levels by suppressing hepatic VLDL production rate, which leads to effective protection against atherosclerosis in the vascular wall.

MicroRNA-21 silencing prolongs islet allograft survival by inhibiting Th17 cells

It has been reported that microRNA-21 (miR-21) augments Th17 responses and contributes to the pathogenesis of autoimmune diseases. Gene knockout or siRNA-induced knockdown of miR-21 in mice resulted in impaired Th17 differentiation and strong resistance to experimental autoimmune encephalomyelitis (EAE). Recently, we validated the miR-21 target IL-10 mRNA and showed that it exerts a pro-inflammatory role by inhibiting IL-10-expressing regulatory B cell (B10) differentiation. The administration of miR-21 antisense oligonucleotides (antagomiR-21) in vivo potently suppressed the severity of EAE, and the suppressive activity was mediated by an increased number of B10 cells. However, the contribution of the miR-21 pathways involved in transplant rejection remains obscure. In this study, we examined the impact of systemic administration of miR-21 inhibitor on allografts in a pancreatic islet transplantation model. We showed that specific miR-21 silencing in vivo significantly prolonged allograft survival (median survival time (MST) 21 days with antagomiR-21 vs 13 days for the control, p < 0.05), and the change was associated with a decrease in Th17 cells (~3-fold) and an increase in B10 cells (~2.4-fold). Moreover, we found that miR-21-silenced B cells mediate this protective role through pro-inflammatory Th17 responses in an IL-10-dependent fashion. Thus, we have revealed a novel mechanistic pathway that modulates Th17 development and alloimmunity. Targeting miR-21 may represent a valuable therapeutic intervention strategy for preventing transplant rejection.

Protective of combination preconditioning on renal ischemia-reperfusion injury in rats

Objective To investigate the effect of Anti TNF-α combined with p38MAPK antisense oligonucleotide on renal funtion and TNF- α, p38MAPK protein expression at different ischemia-reperfusion points. Methods One hundred and twenty normal male Sprague-Dawley rats were divided into 4 groups by simple randomization: sham-operated(sham)group, ischemia-reperfusion(IR)group, Anti TNF-α+ ischemia-reperfusion(Anti TNF-α+ IR)group, Anti TNF-α and p38MAPK antisense oligonucleotide + ischemia-reperfusion(combination preconditioning+ IR) group.Each group comprised 30 rats. Anti TNF-α+ IR group was subjected to ischemia-reperfusion injury with intravenous administration of Anti TNF-α(0.1 mg/kg)5min before reperfusion.Combination preconditioning+ IR group was subjected to ischemia-reperfusion injury with intravenous administration of Anti TNF-α(0.1 mg/kg)and p38MAPK antisense oligonucleotide (5 mg/kg)5 min before reperfusion.IR group with the same injury was followed by saline administration in the same manner.Sham group was subjected to only anesthetization but not to ischemia.Detecting the plasma creatinine and plasma urea nitrogen and two-step inmunohistochemical methods were used to detect the changes of expression of TNF- α, p38MAPK. The measurement data were compared with the t test and the count data were compared with Chi-square test. The date were expressed by (±s). Intergroup comparison translated by variance analysis. Results After reperfusion, the plasma urea nitrogen in the IR group was (15.86±2.41), (21.13±2.21), (25.47±2.29), (30.51±2.03), (35.56±2.47) μmol/L at 0, 1, 3, 6 and 12 h, respectively, and was (25.61±5.40), (32.48±2.30)(68.20±1.20), (84.42±2.43), (96.15±2.23)at 0, 1, 3, 6 and 12 h respectively, and still showed an increasing trend at 12 h. TNF- α mainly expressed in renal proximal convoluted tubules, gradually upregulate with duration of ischemia-reperfusion to 12h of reperfusion. p38MAPK mainly located at distal convoluted tubules, peaked at 6h of reperfusion. But these effects were offset by administration in combination preconditioning group (P<0.05). Conclusion Renal ischemia-reperfusion injury can be alleviated by Anti TNF-α and p38MAPK antisense oligonucleotide treatment. Key words: Reperfusion injury; Kidney; Ischemia; Tumor necrosis factor

Abstract 17101: Targeting of Heparin-Binding EGF-Like Growth Factor (HB-EGF) Effectively Protected Against Hyperlipidemia-Associated Vascular and Hepatic Inflammations

Introduction: Obesity and hepatic VLDL overproduction are key risk factors for the development of dyslipidemia and metabolic inflammatory diseases including atherosclerosis and non-alcoholic fatty liver disease (NAFLD). HB-EGF is a representative mediator for sustained EGFR transactivation under diverse oxidative stress environments including central obesity. A previous report showed that, in C57BL/6 mice, an injection of recombinant HB-EGF significantly increased apoB and TG secretion from the liver; in contrast, HB-EGF antisense oligonucleotide (ASO) administration induced a significant suppression of the hepatic VLDL secretion. Hypothesis: In this study, we tested the hypothesis, which was, that HB-EGF is an autonomous stimulator for the hepatic VLDL production in the liver, which would result in the development of hypertriglyceridemia, causing metabolic inflammatory diseases when under obese and over-nutrition. Methods and Results: In liver-derived cell line HepG2, recombinant HB-EGF treatment induced an autonomous increase of apoB production in the cells. In LDLR deficient mice under a western diet, which showed a list of metabolic syndrome phenotypes, HB-EGF ASO administration induced an effective downregulation of circulatory VLDL and TG level. The HB-EGF ASO administration also showed effective protection against atherosclerosis and non-alcoholic steatohepatitis (NASH) phenotypes in the mouse model. Conclusions: The results suggest that HB-EGF is a positive stimulator of hepatic VLDL production that may cause hypertriglyceridemia under obese and over-nutrition. It was also demonstrated that HB-EGF targeting could be an effective approach to prevent and reverse vascular and hepatic inflammations as shown in a mouse model. Figure: LDLR deficient mice under western diet were treated with control and HB-EGF ASOs for 12 weeks. CD68 positive cell content and inflammatory gene MCP1 transcription level in liver were compared.

Huntingtin suppression restores cognitive function in a mouse model of Huntington's disease.

Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder caused by a mutation in the huntingtin (HTT) protein, resulting in acquisition of toxic functions. Previous studies have shown that lowering mutant HTT has the potential to be broadly beneficial. We previously identified HTT single-nucleotide polymorphisms (SNPs) tightly linked to the HD mutation and developed antisense oligonucleotides (ASOs) targeting HD-SNPs that selectively suppress mutant HTT. We tested allele-specific ASOs in a mouse model of HD. Both early and late treatment reduced cognitive and behavioral impairments in mice. To determine the translational potential of the treatment, we examined the effect of ASO administration on HTT brain expression in nonhuman primates. The treatment induced robust HTT suppression throughout the cortex and limbic system, areas implicated in cognition and psychiatric function. The results suggest that ASOs specifically targeting mutated HTT might have therapeutic effects on HD-mediated cognitive impairments.

Gene Therapy in Mouse Models of Deafness and Balance Dysfunction.

Therapeutic strategies to restore hearing and balance in mouse models of inner ear disease aim to rescue sensory function by gene replacement, augmentation, knock down or knock out. Modalities to achieve therapeutic effects have utilized virus-mediated transfer of wild type genes and small interfering ribonucleic acids; systemic and focal administration of antisense oligonucleotides (ASO) and designer small molecules; and lipid-mediated transfer of Cas 9 ribonucleoprotein (RNP) complexes. This work has established that gene or drug administration to the structurally and functionally immature, early neonatal mouse inner ear prior to hearing onset is a prerequisite for the most robust therapeutic responses. These observations may have significant implications for translating mouse inner ear gene therapies to patients. The human fetus hears by gestational week 19, suggesting that a corollary window of therapeutic efficacy closes early in the second trimester of pregnancy. We hypothesize that fetal therapeutics deployed prior to hearing onset may be the most effective approach to preemptively manage genetic mutations that cause deafness and vestibular dysfunction. We assert that gene therapy studies in higher vertebrate model systems with fetal hearing onset and a comparable acoustic range and sensitivity to that of humans are an essential step to safely and effectively translate murine gene therapies to the clinic.

Inhibition of the extrinsic or intrinsic coagulation pathway during pneumonia-derived sepsis.

Pneumonia is the most frequent cause of sepsis, and Klebsiella pneumoniae is a common pathogen in pneumonia and sepsis. Infection is associated with activation of the coagulation system. Coagulation can be activated by the extrinsic and intrinsic routes, mediated by factor VII (FVII) and factor XII (FXII), respectively. To determine the role of FVII and FXII in the host response during pneumonia-derived sepsis, mice were treated with specific antisense oligonucleotide (ASO) directed at FVII or FXII for 3 wk before infection with K. pneumoniae via the airways. FVII ASO treatment strongly inhibited hepatic FVII mRNA expression, reduced plasma FVII to ~25% of control, and selectively prolonged the prothrombin time. FXII ASO treatment strongly suppressed hepatic FXII mRNA expression, reduced plasma FXII to ~20% of control, and selectively prolonged the activated partial thromboplastin time. Lungs also expressed FVII mRNA, which was not altered by FVII ASO administration. Very low FXII mRNA levels were detected in lungs, which were not modified by FXII ASO treatment. FVII ASO attenuated systemic activation of coagulation but did not influence fibrin deposition in lung tissue. FVII ASO enhanced bacterial loads in lungs and mitigated sepsis-induced distant organ injury. FXII inhibition did not affect any of the host response parameters measured. These results suggest that partial inhibition of FVII, but not of FXII, modifies the host response to gram-negative pneumonia-derived sepsis.

Physiological and druggable skipping of immunoglobulin variable exons in plasma cells.

The error-prone V(D)J recombination process generates considerable amounts of nonproductive immunoglobulin (Ig) pre-mRNAs. We recently demonstrated that aberrant Ig chains lacking variable (V) domains can be produced after nonsense-associated altered splicing (NAS) events. Remarkably, the expression of these truncated Ig polypeptides heightens endoplasmic reticulum stress and shortens plasma cell (PC) lifespan. Many questions remain regarding the molecular mechanisms underlying this new truncated Ig exclusion (TIE-) checkpoint and its restriction to the ultimate stage of B-cell differentiation. To address these issues, we evaluated the extent of NAS of Ig pre-mRNAs using an Ig heavy chain (IgH) knock-in model that allows for uncoupling of V exon skipping from TIE-induced apoptosis. We found high levels of V exon skipping in PCs compared with B cells, and this skipping was correlated with a biallelic boost in IgH transcription during PC differentiation. Chromatin analysis further revealed that the skipped V exon turned into a pseudo-intron. Finally, we showed that hypertranscription of Ig genes facilitated V exon skipping upon passive administration of splice-switching antisense oligonucleotides (ASOs). Thus, V exon skipping is coupled to transcription and increases as PC differentiation proceeds, likely explaining the late occurrence of the TIE-checkpoint and opening new avenues for ASO-mediated strategies in PC disorders.

A serine protease KLK8 emerges as a regulator of regulators in memory: Microtubule protein dependent neuronal morphology and PKA-CREB signaling

The multitude of molecular pathways underlying memory impairment in neurological disorders and aging-related disorders has been a major hurdle against therapeutic targeting. Over the years, neuronal growth promoting factors, intracellular kinases, and specific transcription factors, particularly cyclic AMP response element-binding protein (CREB), have emerged as crucial players of memory storage, and their disruption accompanies many cognitive disabilities. However, a molecular link that can influence these major players and can be a potential recovery target has been elusive. Recent reports suggest that extracellular cues at the synapses might evoke an intracellular signaling cascade and regulate memory function. Herein, we report novel function of an extracellular serine protease, kallikrein 8 (KLK8/Neuropsin) in regulating the expression of microtubule associated dendrite growth marker microtubule-associated protein (MAP2)c, dendrite architecture and protein kinase A (PKA)-CREB signaling. Both knockdown of KLK8 via siRNA transfection in mouse primary hippocampal neurons and via intra-hippocampal administration of KLK8 antisense oligonucleotides in vivo reduced expression of MAP2c, dendrite length, dendrite branching and spine density. The KLK8 mediated MAP2c deficiency in turn inactivated PKA and downstream transcription factor phosphorylated CREB (pCREB), leading to downregulation of memory-linked genes and consequent impaired memory consolidation. These findings revealed a protease associated novel pathway of memory impairment in which KLK8 may act as a “regulator of regulators”, suggesting its exploration as an important therapeutic target of memory disorders.

Investigation into the Mechanism(s) That Leads to Platelet Decreases in Cynomolgus Monkeys During Administration of ISIS 104838, a 2'-MOE-Modified Antisense Oligonucleotide.

ISIS 104838, a 2'-O-methoxyethyl (2'-MOE)-modified antisense oligonucleotide (ASO), causes a moderate, reproducible, dose-dependent, but selflimiting decrease in platelet (PLT) counts in monkeys and humans. To determine the etiology of PLT decrease in cynomolgus monkeys, a 12-week repeat dose toxicology study in 5 cynomolgus monkeys given subcutaneous injections of ISIS 104838 (30-60 mg/kg/week). Monkeys were also injected intravenously with 111Indium(In)-oxine-labeled PLTs to investigate PLT sequestration. In response to continued dosing, PLT counts were decreased by 50%-90% by day 30 in all monkeys. PLT decreases were accompanied by 2- to 4.5-fold increases in immunoglobulin M(IgM), which were typified by a 2- to 5-fold increase in antiplatelet factor 4 (antiPF4) IgM and antiPLT IgM, respectively. Monocyte chemotactic protein 1 increased upon dosing of ISIS 104838, concomitant with a 2- to 6-fold increase in monocyte-derived extracellular vesicles (EVs), indicating monocyte activation but not PLT activation. Despite a 2- to 3-fold increase in von Willebrand factor antigen in all monkeys following ASO administration, only 2 monkeys showed a 2- to 4-fold increase in endothelial EVs. Additionally, a ∼60 - 80%% increase in PLT sequestration in liver and spleen was also observed. Collectively, these results suggest the overall increase in total IgM, antiPLT IgM and/or antiPF4 IgM, in concert with monocyte activation contributed to increased PLT sequestration in spleen and liver, leading to decreased PLTs in peripheral blood.

Gene therapy with antisense oligonucleotides silencing c-myc reduces neointima formation and vessel wall thickness in a mouse model of vein graft disease

Gene therapy for avoiding intimal hyperplasia of vein grafts after coronary artery bypass grafting is still discussed controversially. A promising application of gene therapy in vein grafts is the use of antisense oligonucleotides to block the expression of genes encoding cell cycle regulatory proteins in vascular smooth muscle cells. C-myc, either directly or by regulating the expression of other proteins, controls cell proliferation, apoptosis and cell survival, tissue remodeling, angiogenesis, cell metabolism, production of inflammatory and anti-inflammatory cytokines, and also participates in cell transformation.Forty C57BL/6J mice underwent interposition of the inferior vena cava from isogenic donor mice into the common carotid artery using a previously described cuff technique. Twenty mice received periadventitial administration of antisense oligonucleotides directed against c-myc (treatment group), the other twenty mice received no treatment (control group). All vein grafts were harvested two weeks after surgery, dehydrated, wax embedded, cut into slides of 2 μm thickness, stained and histologically and immunohistochemically examined under light microscope.In our study, we could show the promising effects of antisense oligonucleotide treatment in a mouse model of vein graft disease including the significant reduction of neointimal, media and total vessel wall thickness with a significantly lower percentage of SMA positive cells, elastic fibres and acid mucopolysaccharides in the neointima and media, a decreased vascularization, and a lower expression of PDGFR ß, MMP-9 and VEGF-A positive cells throughout the whole vein graft wall.

Pulmonary Administration of Microparticulate Antisense Oligonucleotide (ASO) for the Treatment of Lung Inflammation.

Targeted delivery to the lung for controlling lung inflammation is an area that we have explored in this study. The purpose was to use microparticles containing an antisense oligonucleotide (ASO) to NF-κB to inhibit the production of proinflammatory cytokines. Microparticles were prepared using the B-290 Buchi Spray Dryer using albumin as the microparticle matrix. Physicochemical characterization of the microparticles showed the size ranged from 2 to 5μm, the charge was - 38.4mV, and they had a sustained release profile over 72h. Uptake of FITC-labeled ASO-loaded microparticles versus FITC-labeled ASO solution by RAW264.7 murine macrophage cells was 5-10-fold higher. After pulmonary delivery of microparticles to Sprague-Dawley rats, the microparticles were uniformly distributed throughout the lung and were retained in the lungs until 48h. Serum cytokine (TNF-α and IL-1β) levels of rats after induction of lung inflammation by lipopolysaccharide were measured until 72h. Animals receiving ASO-loaded microparticles were successful in significantly controlling lung inflammation during this period as compared to animals receiving no treatment. This study was successful in proving that microparticulate ASO therapy was capable of controlling lung inflammation.

Intracerebroventricular Administration of a 2'-O-Methyl Phosphorothioate Antisense Oligonucleotide Results in Activation of the Innate Immune System in Mouse Brain.

Antisense oligonucleotides (AONs) are versatile molecules that can be used to modulate gene expression by binding to RNA. The therapeutic potential of AONs appears particularly high in the central nervous system, due to excellent distribution and uptake in brain cells, as well as good tolerability in clinical trials thus far. Nonetheless, immune stimulation in response to AON treatment in the brain remains a concern. For this reason we performed RNA sequencing analysis of brain tissue from mice treated intracerebroventricularly with phosphorothioate, 2′-O-methyl modified AONs. A significant upregulation of immune system associated genes was observed in brains of AON treated mice, with the striatum showing largest transcriptional changes. Strongest upregulation was seen for the antiviral enzyme 2′-5′-oligoadenylate synthase-like protein 2 (Oasl2) and Bone marrow stromal antigen 2 (Bst2). Histological analysis confirmed activation of microglia and astrocytes in striatum. The upregulation of immune system associated genes was detectable for at least 2 months after the last AON administration, consistent with a continuous immune response to the AON.

Rlip depletion prevents spontaneous neoplasia in TP53 null mice

TP53 (p53) is a tumor suppressor whose functions are lost or altered in most malignancies. p53 homozygous knockout (p53-/-) mice uniformly die of spontaneous malignancy, typically T-cell lymphoma. RALBP1 (RLIP76, Rlip) is a stress-protective, mercapturic acid pathway transporter protein that also functions as a Ral effector involved in clathrin-dependent endocytosis. In stark contrast to p53-/- mice, Rlip-/- mice are highly resistant to carcinogenesis. We report here that partial Rlip deficiency induced by weekly administration of an Rlip-specific phosphorothioate antisense oligonucleotide, R508, strongly inhibited spontaneous as well as benzo(a)pyrene-induced carcinogenesis in p53-/- mice. This treatment effectively prevented large-scale methylomic and transcriptomic abnormalities suggestive of inflammation found in cancer-bearing p53-/- mice. The remarkable efficiency with which Rlip deficiency suppresses spontaneous malignancy in p53-/- mice has not been observed with any previously reported pharmacologic or genetic intervention. These findings are supported by cross-breeding experiments demonstrating that hemizygous Rlip deficiency also reduces the spontaneous malignancy phenotype of p53+/- mice. Rlip is found on the cell surface, and antibodies directed against Rlip were found to inhibit growth and promote apoptosis of cell lines as effectively as Rlip siRNA. The work presented here investigates several features, including oxidative DNA damage of the Rlip-p53 association in malignant transformation, and offers a paradigm for the mechanisms of tumor suppression by p53 and the prospects of suppressing spontaneous malignancy in hereditary cancer syndromes such as Li-Fraumeni.

Identification and Rescue of Splice Defects Caused by Two Neighboring Deep-Intronic ABCA4 Mutations Underlying Stargardt Disease

Sequence analysis of the coding regions and splice site sequences in inherited retinal diseases is not able to uncover ∼40% of the causal variants. Whole-genome sequencing can identify most of the non-coding variants, but their interpretation is still very challenging, in particular when the relevant gene is expressed in a tissue-specific manner. Deep-intronic variants in ABCA4 have been associated with autosomal-recessive Stargardt disease (STGD1), but the exact pathogenic mechanism is unknown. By generating photoreceptor precursor cells (PPCs) from fibroblasts obtained from individuals with STGD1, we demonstrated that two neighboring deep-intronic ABCA4 variants (c.4539+2001G>A and c.4539+2028C>T) result in a retina-specific 345-nt pseudoexon insertion (predicted protein change: p.Arg1514Leufs∗36), likely due to the creation of exonic enhancers. Administration of antisense oligonucleotides (AONs) targeting the 345-nt pseudoexon can significantly rescue the splicing defect observed in PPCs of two individuals with these mutations. Intriguingly, an AON that is complementary to c.4539+2001G>A rescued the splicing defect only in PPCs derived from an individual with STGD1 with this but not the other mutation, demonstrating the high specificity of AONs. In addition, a single AON molecule rescued splicing defects associated with different neighboring mutations, thereby providing new strategies for the treatment of persons with STGD1. As many genes associated with human genetic conditions are expressed in specific tissues and pre-mRNA splicing may also rely on organ-specific factors, our approach to investigate and treat splicing variants using differentiated cells derived from individuals with STGD1 can be applied to any tissue of interest.

Smad7 positively regulates keratinocyte proliferation in psoriasis.

Transforming growth factor (TGF)-β1 exerts inhibitory effects on keratinocyte proliferation. To examine whether Smad7, a known inhibitor of TGF-β1 signalling, is involved in psoriasis-associated keratinocyte hyperproliferation. Smad7 was evaluated in skin sections of patients with psoriasis and healthy controls and in mice with Aldara-induced skin pathology by real-time polymerase chain reaction and immunohistochemistry. To assess whether Smad7 positively regulates in vivo keratinocyte growth, mice treated with Aldara received daily cutaneous administration of Smad7 antisense oligonucleotide (AS). Keratin (K)6 and K16, cell-cycle-associated factors, cell-cycle and cell proliferation were evaluated in HaCaT cells either treated with Smad7 AS or transfected with Smad7 plasmid and in mice given Smad7 AS. Smad7 was highly expressed in keratinocytes of patients with psoriasis and of mice treated with Aldara. In HaCaT cells, Smad7 knockdown inhibited cell growth, reduced K6 and K16 expression and promoted accumulation of cells in the S-phase of the cell cycle. Smad7-deficient keratinocytes exhibited reduced levels of CDC25A protein, a phosphatase that facilitates progression of cells through the S-phase, and hyperphosphorylation of eukaryotic initiation factor 2 (eIF2)α, a negative regulator of CDC25 protein translation. Consistently, Smad7 overexpression in HaCaT cells was followed by induction of K6 and K16 and increased cell proliferation. Topical application of Smad7 AS to Aldara-treated mice reduced epidermal thickness. Our data show that Smad7 is overexpressed in human and murine psoriasis and suggest a key role of this molecule in the control of keratinocyte proliferation.