Generation Antisense Oligonucleotide Research Articles

Next Generation Exon 51 Skipping Antisense Oligonucleotides for Duchenne Muscular Dystrophy.

In the last two decades, antisense oligonucleotides (AONs) that induce corrective exon skipping have matured as promising therapies aimed at tackling the dystrophin deficiency that underlies the severe and progressive muscle fiber degeneration in Duchenne muscular dystrophy (DMD) patients. Pioneering first generation exon 51 skipping AONs like drisapersen and eteplirsen have more recently been followed up by AONs for exons 53 and 45, with, to date, a total of four exon skipping AON drugs having reached (conditional) regulatory US Food and Drug Administration (FDA) approval for DMD. Nonetheless, considering the limited efficacy of these drugs, there is room for improvement. The aim of this study was to develop more efficient [2'-O-methyl-modified phosphorothioate (2'OMePS) RNA] AONs for DMD exon 51 skipping by implementing precision chemistry as well as identifying a more potent target binding site. More than a hundred AONs were screened in muscle cell cultures, followed by a selective comparison in the hDMD and hDMDdel52/mdx mouse models. Incorporation of 5-methylcytosine and position-specific locked nucleic acids in AONs targeting the drisapersen/eteplirsen binding site resulted in 15-fold higher exon 51 skipping levels compared to drisapersen in hDMDdel52/mdx mice. However, with similarly modified AONs targeting an alternative site in exon 51, 65-fold higher skipping levels were obtained, restoring dystrophin up to 30% of healthy control. Targeting both sites in exon 51 with a single AON further increased exon skipping (100-fold over drisapersen) and dystrophin (up to 40%) levels. These dystrophin levels allowed for normalization of creatine kinase (CK) and lactate dehydrogenase (LDH) levels, and improved motor function in hDMDdel52/mdx mice. As no major safety observation was obtained, the improved therapeutic index of these next generation AONs is encouraging for further (pre)clinical development.

Rise and fall of fomivirsen, the first approved gene silencing medicine : A historical review

Fomivirsent was approved by the FDA in 1998 and by the EMA in 1999 as the very first antisense drug used to treat CMV retinitis in patients with AIDS. To date, it has been the only first generation antisense oligonucleotide used in therapy. Fomivirsen has been a pioneer in this field and has demonstrated the usefulness of the antisense tehcnology for medicinal science. However, after three years of use, fomivirsen has been withdrawn from the market (in the US in 2001 and in the EU in 2002), and nowadays, gene silencing drugs with a more advanced chemical structure and more complex mechanism of action are used in medicine. On the occasion of the 20th anniversary of its European withdrawal, we briefly overview the history of fomivirsen.

Towards next generation antisense oligonucleotides: mesylphosphoramidate modification improves therapeutic index and duration of effect of gapmer antisense oligonucleotides.

The PS modification enhances the nuclease stability and protein binding properties of gapmer antisense oligonucleotides (ASOs) and is one of very few modifications that support RNaseH1 activity. We evaluated the effect of introducing stereorandom and chiral mesyl-phosphoramidate (MsPA) linkages in the DNA gap and flanks of gapmer PS ASOs and characterized the effect of these linkages on RNA-binding, nuclease stability, protein binding, pro-inflammatory profile, antisense activity and toxicity in cells and in mice. We show that all PS linkages in a gapmer ASO can be replaced with MsPA without compromising chemical stability and RNA binding affinity but these designs reduced activity. However, replacing up to 5 PS in the gap with MsPA was well tolerated and replacing specific PS linkages at appropriate locations was able to greatly reduce both immune stimulation and cytotoxicity. The improved nuclease stability of MsPA over PS translated to significant improvement in the duration of ASO action in mice which was comparable to that of enhanced stabilized siRNA designs. Our work highlights the combination of PS and MsPA linkages as a next generation chemical platform for identifying ASO drugs with improved potency and therapeutic index, reduced pro-inflammatory effects and extended duration of effect.

Effect of inclisiran on haematological and immunological biomarkers: A Pooled analysis of ORION-9, -10 and -11 trial data

Background and Aims: Haematological and immunological adverse effects and significant injection-site adverse events (AEs) have been reported with some early generation anti-sense oligonucleotide (RNA-based) therapies, which required frequent dosing and had non-specific organ uptake. Inclisiran is a novel small interfering RNA (siRNA) therapy with twice-yearly dosing, and conjugated with trivalent N-Acetylgalactosamine (GalNAc) enabling targeted hepatic delivery. Here, we assessed the effects of inclisiran on haematological and immunological biomarkers.

Abstract ND11: The discovery and characterization of ION-537: A next generation antisense oligonucleotide inhibitor of YAP1 in preclinical cancer models

Abstract The tumor suppressive Hippo pathway is frequently dysregulated in cancer, resulting in the constitutive activation of YAP1 in a variety of solid cancers including hepatocellular cancer (HCC) and head and neck squamous cell carcinoma (HNSCC) among others. Here, we identified potent next-generation constrained ethyl (cEt) antisense oligonucleotides (ASOs) to selectively target either human or mouse YAP1 and evaluated these in several preclinical models of both HCC and HNSCC. In the genetically engineered mouse (GEM) model of HCC (Salvador KO mice) and in the carcinogen-induced models of HCC (diethylnitrosamine, DEN-induced HCC) systemic treatment with mouse YAP1 ASOs resulted in a marked reduction in YAP1 protein level in (~90% reduction) and strong regressions of established tumors. Interestingly, YAP1 ASO treatment also resulted in significant infiltration of T cells and myeloid lineage cells in the tumors in these immune-competent mouse models of HCC suggesting a role in immune activation. Consistent with this notion, YAP1 ASO treatment in combination with anti-PD1 antibody treatment led to enhanced antitumor activity. In HNSCC, loss of the tumor suppressor FAT1 is a frequent occurrence and results in activation and nuclear localization of YAP1. In a panel of human HNSCC cell lines, proliferation was strongly reduced by human YAP1 ASOs selectively in the cell lines harboring homozygous copy loss or nonsense mutations of FAT1 while minimal antiproliferative activity was observed in cells with wild type FAT1. Importantly, the nuclear localization of YAP1 appeared to be a key determining factor for the sensitivity of human HNSCC cells to YAP1 depletion by ASO. In vivo treatment of mice bearing human CAL33 tumors that harbor FAT1 mutation, YAP1 ASOs greatly reduced YAP1 expression in tumors and suppressed tumor growth in both subcutaneous and orthotopic models. Collectively, these results suggest a potential therapeutic use of YAP1 ASOs in the treatment of both HCC and HNSCC with YAP1 activation as a single agent or in combination with immuno-oncology drugs. Based on these finding broad human ASO clinical candidates screens were performed leading to the selection of ION-537, a potent ASO drug targeting human YAP1 has now advanced into human clinical trials in cancer patients with YAP1 activation. Citation Format: A. Robert Macleod. The discovery and characterization of ION-537: A next generation antisense oligonucleotide inhibitor of YAP1 in preclinical cancer models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr ND11.

Therapeutic Targeting of Interferon Regulatory Factor 4 with Next Generation Antisense Oligonucleotides Produces Robust In Vivo Antitumor Activity in Preclinical Models of Multiple Myeloma

Despite significant recent advances in therapeutic options, multiple myeloma (MM) remains an incurable disease, and novel agents targeting novel molecular pathways with activity in advanced patients are urgently needed. Interferon Regulatory Factor 4 (IRF4) is a transcription factor involved in immune responses in normal B and T cells, and is strongly implicated in the development of hematological malignancies, especially MM. Although recent publications have pointed to IRF4 as a driver oncogene in the progression of MM, it is considered an intractable target by conventional therapeutic approaches.Here we describe an antisense oligonucleotide (ASO) approach to selectively target IRF4. Administration of next generation human IRF4 ASOs via ‘free-uptake’ (without transfection) to a large panel of human MM cell lines resulted in a dose-dependent IRF4 reduction with concomitant induction of apoptosis. ASO-mediated IRF4 depletion also decreased the levels of c-Myc, another key oncogene in MM. MM cells were sensitive to even modest depletion of IRF4 (<50%), suggesting the strong dependence of MM cells on IRF4 for their survival. IRF4 ASOs also enhanced the sensitivity of the tumor cells to the treatment of IMiD (lenalidomide), proteasome inhibitor (Bortezomib) and BTK inhibitor (Ibrutinib) in MM cell, including KMS-11, JJN3 and MM1.R. In addition, Bortezomib-resistant clones of KMS-11 cells established from a long-term exposure to the drug remained sensitive to IRF4 inhibition. Importantly, in two different subcutaneously implanted MM tumor models, systemic administration of 2 independent human IRF4 ASOs depleted IRF4 protein levels in tumors and inhibited the growth of tumors compared to control treatment. Moreover, in a clinically relevant MM mouse model where human MM cells accumulate in the mouse bone marrow, treatment with human IRF4 ASOs substantially prolonged the survival of the tumor-bearing animals along with specific downregulation of human IRF4 and its downstream gene c-Myc. Finally, systematic treatment of mice with mouse-selective IRF4 ASOs at 50 mg/kg, twice a week for 4 weeks was well tolerated in normal mice, which is consistent with the lack of phenotypes observed in IRF4 heterozygous animals. Taken together, these results suggest that MM cells rely heavily on the survival pathway mediated by IRF4 and that selective knockdown of IRF4 by next generation ASOs is an attractive therapeutic strategy for the treatment of patients with advanced treatment-refractory MM. DisclosuresZhou:Ionis Pharmaceuticals: Employment, Equity Ownership. Schmidt:Ionis Pharmaceuticals: Employment. Kim:Ionis Pharmaceuticals: Employment. MacLeod:Ionis Pharmaceuticals: Employment.

Pharmacokinetics and Pharmacodynamics of Ionis-FXIRx, an Antisense Inhibitor of Factor XI, in Patients with End-Stage Renal Disease on Hemodialysis

BACKGROUNDPatients with end-stage renal disease (ESRD) on hemodialysis are at high risk of both thrombotic and bleeding events. Experimental and clinical data indicate that reducing factor XI (FXI), a key component of the intrinsic pathway, prevents thrombosis without causing bleeding. FXI activity can be lowered with IONIS-FXIRx, a 2nd generation antisense oligonucleotide that specifically reduces human FXI mRNA expression in the liver. This study sought to determine the safety, pharmacokinetics (PK) and pharmacodynamics (PD) of IONIS-FXIRx in participants with ESRD. METHODSIn this Phase 2 multicenter study, we enrolled a total of 49 ESRD patients: (A) 6 participants receiving chronic in-center hemodialysis (HD) to receive an open-label single-dose of 300 mg IONIS-FXIRx either 5 minutes before or 10 minutes after HD and assessed the effects of HD on peak (Cmax) and the extent of exposure (AUC0-24hr) parameters of IONIS-FXIRx and (B) another 43 participants in a double-blind design to one of two multiple-dose regimens (200 mg or 300 mg) IONIS-FXIRx or placebo for 12 weeks with 12 weeks of additional follow up. In the double-blind portion, IONIS-FXIRx was administered subcutaneously ~10 minutes after HD. For PK, plasma trough concentration and apparent elimination half-life (t1/2λz) were evaluated. The PD parameters including FXI activity and antigen, aPTT, prothrombin time (PT), and INR were evaluated in the per-protocol population (N= 34) that was defined as all participants who received the complete protocol-specified administration of IONIS-FXIRx or placebo through week 8 and who did not have any major protocol violations. The rate and frequency of clotting on the HD filters and circuit were qualitatively measured with a clotting scale as an exploratory PD analysis. RESULTSThe PK cohort included 6 males (5 white) with a mean age of 61 years. The plasma PK profile of IONIS-FXIRx was not significantly altered whether injected before or after HD. After multiple dosing, plasma Cmax values were dose-dependent and similar between Day 1 (first dose) and Day 78 (last dose), suggesting no accumulation of IONIS-FXIRx in ESRD participants after 12-weeks of dosing. After treatment cessation, IONIS-FXIRx had an apparent elimination half-life of approximately 2 weeks. In the double-blind portion of the study, 22 (51.2%) of the participants were male, 24 (55.8%) were white and the mean age was 59 years. Attenuation of FXI activity was associated with a reduction in the incidence of severe clotting events on both the air filter and dialyzer (Table). The reduction in severe clotting events was observed when FXI activity was < 0.4 U/mL. Consistent with prior observations in healthy subjects, IONIS-FXIRx prolonged aPTT in a time and dose-dependent manner with maximum mean percent changes from baseline of 53.3% (p < 0.05) for the 200 mg dose group and 75.6% (p < 0.05) for the 300 mg dose group at Week 14, but had no effect on PT. Treatment emergent adverse events (TEAEs) were mostly mild; participants reporting serious TEAEs were 1 (16.7%) for the PK cohort, 4 (30.8%) for placebo and 3 (20.0%) each for the 200 mg and 300 mg IONIS-FXIRx dose groups. There were no drug-related SAEs and no drug-related major or clinically-relevant non-major bleeding events. Minor bleeds were primarily observed in the 300 mg multiple-dose group at AV fistula/graft sites but did not correlate with reduction of FXI activity. No clinically significant reductions in platelet counts were reported during the study. CONCLUSIONSThis study demonstrated that (i) HD had no effect on IONIS-FXIRx PK, (ii) IONIS-FXIRx was well tolerated and produced sustained, and dose-dependent reductions in FXI antigen and activity, and (iii) IONIS-FXIRx reduced severe dialysis circuit clotting events beyond standard heparin use. Collectively, these data support further evaluation of IONIS-FXIRx as a potentially safe, effective antithrombotic therapy in ESRD patients on HD. [Display omitted] DisclosuresBethune:Ionis Pharmaceuticals: Employment. Walsh:Population Health Research Institute: Employment; Ionis Pharmaceuticals: Consultancy. Jung:Ionis Pharmaceuticals: Employment. Yu:Ionis Pharmaceuticals: Employment. Geary:Ionis Pharmaceuticals: Employment. Bhanot:Ionis Pharmaceuticals: Employment.

In Vivo Administration of Therapeutic Antisense Oligonucleotides.

With the rapid revolution in RNA/DNA sequencing technologies, it is evident that mammalian genomes express tens of thousands of long noncoding RNAs (lncRNAs). Since a large majority of lncRNAs have been functionally implicated in cancer development and progression, there is an increasing appreciation for the use of antisense oligonucleotide (ASO)-based therapies targeting lncRNAs in several cancers. Despite their great potential in therapeutic applications, their use is still limited due to cellular toxicity and shortcomings in achieving required stability in biological fluids and tissue uptake. To overcome these limitations, major changes in ASO chemistry have been introduced to generate second and third generation ASOs, including locked nucleic acids (LNA) technology. Here we describe two different LNA-ASO delivery approaches, a peritumoral administration and a systemic delivery in xenograft models of lung adenocarcinoma, that significantly reduced tumor growth without inducing toxicity.

Antisense oligonucleotides extend survival and reverse decrement in muscle response in ALS models.

Mutations in superoxide dismutase 1 (SOD1) are responsible for 20% of familial ALS. Given the gain of toxic function in this dominantly inherited disease, lowering SOD1 mRNA and protein is predicted to provide therapeutic benefit. An early generation antisense oligonucleotide (ASO) targeting SOD1 was identified and tested in a phase I human clinical trial, based on modest protection in animal models of SOD1 ALS. Although the clinical trial provided encouraging safety data, the drug was not advanced because there was progress in designing other, more potent ASOs for CNS application. We have developed next-generation SOD1 ASOs that more potently reduce SOD1 mRNA and protein and extend survival by more than 50 days in SOD1G93A rats and by almost 40 days in SOD1G93A mice. We demonstrated that the initial loss of compound muscle action potential in SOD1G93A mice is reversed after a single dose of SOD1 ASO. Furthermore, increases in serum phospho-neurofilament heavy chain levels, a promising biomarker for ALS, are stopped by SOD1 ASO therapy. These results define a highly potent, new SOD1 ASO ready for human clinical trial and suggest that at least some components of muscle response can be reversed by therapy.

In vivo Safety and Efficacy of a 3rd generation Antisense Oligonucleotide targetting TGF-ßRII to treat Amyotrophic Lateralsclerosis (P1.325)

In vivo Safety and Efficacy of a 3rd generation Antisense Oligonucleotide targetting TGF-ßRII to treat Amyotrophic Lateralsclerosis (P1.325)

Endosomal Escape of ASOs Internalized by Stabilin Receptors Is Regulated by Rab5C and EEA1.

One of the major drug classes that has been gaining traction in the pharmacological world has been the emergence of Antisense oligonucleotides (ASOs). Increased modifications of these drugs have led to major breakthroughs leading to a boost in this drug class being adapted to clinical trials. Second generation (Gen 2) Antisense oligonucleotides show increased nuclease stability and affinity for their RNA targets which has translated to improved potency and therapeutic index in the clinic. Gen 2 ASOs are typically modified using the phosphorothioate (PS) backbone modification which enhances ASO interactions with plasma, cell surface and intracellular proteins. This facilitates ASO distribution to peripheral tissues and also promotes cellular uptake after injection into animals. Recent work of ours has shown that a group of class H Stabilin receptors specifically internalize PS‐ASOs in the sinusoidal cells of the liver and the spleen. This work identifies a breakthrough candidate for the determination of a molecular pathway for these drugs to reach their desired target in the cell. Further exploration of the endocytotic uptake by these proteins has led to a rudimentary understanding of this pathway. By modulating expression of specific proteins involved in the trafficking and maturation of the endo‐lysosomal compartments, we show that the proteins Rab5C and EEA1 play a direct role in the early endosomal pathway. These proteins, along with Rab7A and LBPA (lyso‐lipid) in the late endosomal pathway, are important for trafficking of PS‐ASOs and facilitate their escape from endo‐lysosomal compartments after Stabilin‐mediated internalization. In conclusion, this research identifies key interacting partners involved in ASO‐containing endosomal maturation, as well as late endosomal partners involved in PS‐ASO endosomal release. This work also identifies the limiting proteins in the pathway for PS‐ASO translocation and escape from the endosome.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Oligonucleotide Therapies for the Lung: Ready to Return to the Clinic?

The most promising targets for treating lung diseases are often intracellular molecules recalcitrant to conventional pharmacotherapy. In this respect, antisense oligonucleotides (ASO) would be valuable tools to treat airways disease, as the plurality of their potential mechanisms of action is well-established, their efficiency has been extensively validated in-vitro, and their potential clinical value has been demonstrated with numerous regulatory approvals. Yet in spite of high confidence in the treatment rationale and mechanism of action, it appears that oligonucleotides delivered topically to the lung either rapidly access circulation via epithelial transcytosis or are removed by alveolar macrophages, exerting minimal if any action in the cytosol of cells relevant to lung disease. Moreover, use of cell penetrating peptides, liposomes or nanoparticle delivery systems has not so far been shown to eliminate circulatory clearance, may activate immune responses, or drive macrophage phenotypic changes that, together or in isolation, may present risks to patients.

Abstract 1582: Therapeutic potential of combination therapy using a next generation antisense oligonucleotide targeting the androgen receptor and AKT inhibition with AZD5363 in genetically engineered mouse models of prostate cancer

Abstract Prostate cancer is highly dependent on androgen receptor (AR) and PI3K/AKT signaling pathways for survival and disease progression. Preclinical evidence suggests that combinatorial approaches targeting both AR and PI3K/AKT activity improves treatment efficacy. However, sustained responses from traditional and next-generation anti-androgen therapies targeting AR remain elusive in clinical practice due to inherent/acquired resistance resulting in lethal castration-resistant prostate cancer (CRPC). Mechanisms for continued AR transcriptional activity may be ligand dependent or independent but still require AR gene expression. Persistent AR gene expression is a key feature of CRPC. Thus, blocking AR gene expression by antisense oligonucleotides (ASO) is a logical approach to CRPC. We previously showed that monotherapy with ISIS581088, a generation 2.5 ASO targeting mouse AR, demonstrated strong antitumor activity in a transgenic mouse model of PTEN-deficient prostate cancer. In this study we show the antitumor effects of combined therapy of ISIS581088 and AZD5363, a potent AKT inhibitor and demonstrate the therapeutic benefit of combination therapy in a clinically relevant mouse model of CRPC. Sixteen-week-old mice with PTEN-/- castration-naïve prostate tumors were treated with ISIS581088 and AZD5363 alone or in combination for four weeks. Tumor growth inhibition rates were 41.2%, 20.2% and 54.4% for ISIS581088, AZD5363 and ISIS581088/AZD5363 treatment groups, respectively, P&amp;lt;0.001. In a model of mouse CRPC, 16-week-old mice with PTEN-/- castration-resistant prostate tumors (eight weeks post castration) experienced reduced tumor burden with all treatments but no enhancement was observed when the compounds were administered in combination compared with monotherapy drug treatments. In a randomized trial of advanced CRPC in PTEN/P53 double knockout mice, combination therapy significantly increased overall survival. Median survival were 18, 17 21, 22, and 38 days for control vehicle, control ASO, ISIS581088, AZD5363 and ISIS581088/AZD5363 treatment groups, respectively, P=0.041. In conclusion, our data shows that combination therapy significantly reduced tumor burden in mice with castration-naïve tumors compared to those treated with monotherapy. Notably, combination therapy did not produce an additive effect in an early stage CRPC intervention model. Still, combination therapy demonstrated a clear advantage in prolonging overall survival in a long-term randomized mouse trial of PTEN/P53-deficient CRPC. Thus, our data provides preclinical evidence to support that next generation ASOs targeting AR in combination with AKT inhibition is a potentially effective treatment approach for CRPC. Citation Format: Marco A. De Velasco, Yurie Kura, Naomi Ando, Koichi Sugimoto, Kazuko Sakai, Barry R. Davies, Youngsoo Kim, A. Robert MacLeod, Masahiro Nozawa, Kazuhiro Yoshimura, Kazuhiro Yoshikawa, Kazuto Nishio , Hirotsugu Uemura. Therapeutic potential of combination therapy using a next generation antisense oligonucleotide targeting the androgen receptor and AKT inhibition with AZD5363 in genetically engineered mouse models of prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1582. doi:10.1158/1538-7445.AM2017-1582

Abstract 3684: Inhibition of STAT3 by antisense oligonucleotide treatment decreases the immune suppressive tumor microenvironment in syngeneic and GEM tumor models

Abstract AZD9150, a gen2.5 antisense oligonucleotide (ASO) targeting human STAT3, has improved drug-like properties compared to previous generation ASO therapeutics, including increased stability and resistance to nucleases, reduced proinflammatory effects, and enhanced potency. We have previously reported that in tumors, STAT3 ASOs are taken up preferentially in stromal and immune cells of the tumor microenvironment (TME). Since AZD9150 is selective for human STAT3, we used a surrogate ASO (muSTAT3 ASO) to explore the pharmacodynamics of ASO-mediated STAT3 inhibition in syngeneic and genetically engineered mouse (GEM) tumor models, focusing on effects in the TME. In mice bearing subcutaneous CT-26 tumors, treatment with muSTAT3 ASO at 50 mg/kg, s.c., on a qdx5/wk schedule decreased STAT3 levels in immune cell subsets in the tumor and in circulating leukocytes by 40 - 60%, similar to the decrease in STAT3 achievable in circulating leukocytes in human patients after AZD9150 treatment. In a Nanostring analysis (nCounter mouse immunology panel) of CT-26 tumors from muSTAT3 ASO treated mice, CD163 (M2 immune suppressive macrophage marker) was the gene most consistently and significantly downregulated, by an average of 84% in three independent experiments, and was confirmed by immunohistochemistry (IHC). Flow cytometry analysis of myeloid subpopulations - tumor associated macrophages (F4/80+ TAMs), monocytic myeloid derived suppressor cells, and granulocytic cells - showed a decrease in TAMs averaging 69% across three independent experiments. The analysis was extended to include IHC for arginase (Arg, a marker of functional immune suppression activity). Subpopulations of cells identified included Arg+, CD163+, and Arg+CD163+. Treatment with muSTAT3 ASO decreased these populations by 79%, 88% and 97% respectively, compared to control treatment. These populations were also analyzed in two GEM tumor models - the KPC pancreatic cancer model, and a PTEN -/- prostate cancer model - which have a TME more representative of that found in tumors in the clinic. While the specific changes varied across the models, likely reflecting differences in TME makeup, a reduction in immune suppressive cell populations was present in both GEM models, including a decrease in CD163+ cells of 79% (along with modest antitumor activity) in the PTEN -/- prostate model after muSTAT3 ASO treatment. These results indicate that selective STAT3 inhibition can reduce immune suppressive cell populations in the TME, and suggest that STAT3 inhibition has the potential to enhance the antitumor activity of T-cell targeted therapies, such as those targeting the PD1-PDL1 axis. In support of this hypothesis, we observed that addition of muSTAT3 ASO to anti-PD-L1 Ab treatment significantly enhanced the antitumor activity of PD-L1 Ab treatment in two subcutaneous syngeneic tumor models, CT-26 and A20. Citation Format: Rich Woessner, Vasu Sah, Patricia McCoon, Shaun Grosskurth, Nanhua Deng, Rachel DuPont, Deborah Lawson, Lourdes Pablo, Corinne Reimer, Marco A. De Velasco, Hirotsugu Uemura, Juliana Candido, Paul Lyne. Inhibition of STAT3 by antisense oligonucleotide treatment decreases the immune suppressive tumor microenvironment in syngeneic and GEM tumor models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3684. doi:10.1158/1538-7445.AM2017-3684

Electronic Structures of LNA Phosphorothioate Oligonucleotides.

Important oligonucleotides in anti-sense research have been investigated in silico and experimentally. This involves quantum mechanical (QM) calculations and chromatography experiments on locked nucleic acid (LNA) phosphorothioate (PS) oligonucleotides. iso-potential electrostatic surfaces are essential in this study and have been calculated from the wave functions derived from the QM calculations that provide binding information and other properties of these molecules. The QM calculations give details of the electronic structures in terms of e.g., energy and bonding, which make them distinguish or differentiate between the individual PS diastereoisomers determined by the position of sulfur atoms. Rules are derived from the electronic calculations of these molecules and include the effects of the phosphorothioate chirality and formation of electrostatic potential surfaces. Physical and electrochemical descriptors of the PS oligonucleotides are compared to the experiments in which chiral states on these molecules can be distinguished. The calculations demonstrate that electronic structure, electrostatic potential, and topology are highly sensitive to single PS configuration changes and can give a lead to understanding the activity of the molecules.

Abstract 227: Megalin Regulates Angiotensinogen and Contributes to Atherosclerosis

Objective: Angiotensinogen (AGT) is the only substrate of the renin-angiotensin system to generate angiotensin peptides, including angiotensin II (AngII), a critical contributor to atherosclerosis. AGT interacts with megalin in proximal convoluted tubules of kidney. The purpose of this study was to determine effects of megalin on AGT, AngII, and atherosclerosis in mice. Methods and Results: Male C57BL/6 mice were injected subcutaneously with vehicle (PBS) or megalin second generation antisense oligonucleotides (ASO) for 5 weeks. Inhibition of megalin was confirmed by more than 80% reduction of megalin mRNA in kidney. Urine was collected using metabolic cages after 5 weeks of vehicle or ASO injections. At termination, blood was collected with EDTA and protease inhibitor cocktail to measure plasma concentrations of renin and AngII, respectively. Urine AGT and renin concentrations were profoundly increased, accompanied by reduction of renal, but not plasma, AngII production. To determine whether megalin inhibition influences atherosclerosis, male LDL receptor -/- mice were injected with vehicle, control ASO, or megalin ASO for 13 weeks. Western diet was started 1 week after the first injection and continued for 12 weeks. Atherosclerosis was quantified by en face analysis of the aortic intimal surface from the ascending aorta to 3 mm proximal to the left subclavian artery branch. Megalin ASO administration led to more than 70% reductions of megalin mRNA in kidney. Consistent with the effects in C57BL/6 mice, AGT concentrations were significantly higher in urine (Vehicle, control ASO and megalin ASO groups: 85 ± 10, 109 ± 71, and 4616 ± 637 ng/ml, respectively; P&lt;0.001 by one way ANOVA with Holm-Sidak method). Plasma total cholesterol concentrations did not differ among groups. Megalin inhibition reduced atherosclerotic lesion area compared to the other two groups (Percent lesion area in Vehicle, control ASO and megalin ASO groups: 23 ± 2, 20 ± 2, 12 ± 1 %, respectively; P &lt; 0.001 by one way ANOVA with Holm-Sidak method). Conclusions: Inhibition of megalin increased urine AGT and renin, reduced renal AngII concentrations, and diminished hypercholesterolemia-induced atherosclerosis in mice.

Antisense Oligonucleotide-Based Therapy for Neuromuscular Disease.

Neuromuscular disorders such as Duchenne Muscular Dystrophy and Spinal Muscular Atrophy are neurodegenerative genetic diseases characterized primarily by muscle weakness and wasting. Until recently there were no effective therapies for these conditions, but antisense oligonucleotides, a new class of synthetic single stranded molecules of nucleic acids, have demonstrated promising experimental results and are at different stages of regulatory approval. The antisense oligonucleotides can modulate the protein expression via targeting hnRNAs or mRNAs and inducing interference with splicing, mRNA degradation, or arrest of translation, finally, resulting in rescue or reduction of the target protein expression. Different classes of antisense oligonucleotides are being tested in several clinical trials, and limitations of their clinical efficacy and toxicity have been reported for some of these compounds, while more encouraging results have supported the development of others. New generation antisense oligonucleotides are also being tested in preclinical models together with specific delivery systems that could allow some of the limitations of current antisense oligonucleotides to be overcome, to improve the cell penetration, to achieve more robust target engagement, and hopefully also be associated with acceptable toxicity. This review article describes the chemical properties and molecular mechanisms of action of the antisense oligonucleotides and the therapeutic implications these compounds have in neuromuscular diseases. Current strategies and carrier systems available for the oligonucleotides delivery will be also described to provide an overview on the past, present and future of these appealing molecules.

Abstract LB-238: Direct targeting of oncogenic transcription factors using next-generation oligonucleotides as a novel therapeutic strategy for translocation-positive sarcomas

Abstract Many sarcoma subtypes including Ewing sarcoma (ES), alveolar rhabdomyosarcoma (ARMS), synovial sarcoma (SS), and desmoplastic small round cell tumor (DSRCT), are driven by oncogenic chimeric transcription factors. Outcomes for these diseases remain unacceptably poor despite intensification of standard therapies. Although these translocation positive sarcomas possess well-defined oncogenic drivers, as transcription factors that lack intrinsic enzymatic activity, they have been considered undruggable. We have developed a two-tiered pre-clinical therapeutic program 1) creating and characterizing faithful in vitro and in vivo models of these rare malignancies and 2) designing next generation antisense oligonucleotides (ASOs) to directly target the oncogenic chromosomal translocations underlying these multiple sarcoma subtypes. As proof of principle, we present here the program experience with DSRCT, a rare sarcoma for which there has been an overwhelming paucity of both biological tools for pre-clinical investigation, as well as clinical therapeutic options. We have extensively characterized two new DSRCT cell lines harboring the characteristic oncogenic EWSR1-WT1 t(11;22)(p13;q12) fusion, and have used these tools to develop orthotopic xenograft models. Utilizing these accurate model systems we have designed and evaluated a series of ASO compounds directly targeting the EWSR1-WT1 translocation product. Among the series of ASOs developed, we identified D2A, a selective and potent inhibitor of EWSR1-WT1. In vitro, we have shown D2A to abolish the EWSR1-WT1 fusion product with exquisite specificity in a dose-dependent fashion. D2A similarly exhibits efficacy in vivo, with anti-tumorigenic activity in our pre-clinical models resulting in an 80% reduction in xenograft tumor growth. With the aid of authentic in vitro and in vivo modeling systems, this ASO platform has wide-ranging implications for both biological as well as therapeutic advances. Citation Format: Emily Slotkin, Lee Spraggon, Julija Hmeljak, Luciano Martelotto, Romel Somwar, Elisa de Stanchina, Luca Cartegni, Marc Ladanyi. Direct targeting of oncogenic transcription factors using next-generation oligonucleotides as a novel therapeutic strategy for translocation-positive sarcomas. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-238.

MP13-20 SECOND-GENERATION ANTISENSE OLIGONUCLEOTIDE - HSP27 A NEW INTRAVESICAL TREATMENT FOR BLADDER CANCER: PHASE 1CLINICAL TRIAL.

You have accessJournal of UrologyBladder Cancer: Non-Invasive I1 Apr 2016MP13-20 SECOND-GENERATION ANTISENSE OLIGONUCLEOTIDE - HSP27 A NEW INTRAVESICAL TREATMENT FOR BLADDER CANCER: PHASE 1CLINICAL TRIAL. Sebastian Frees, Eliana Beraldi, Kim Chi, Ladan Fazli, Peter Black, Martin Gleave, and Alan So Sebastian FreesSebastian Frees More articles by this author , Eliana BeraldiEliana Beraldi More articles by this author , Kim ChiKim Chi More articles by this author , Ladan FazliLadan Fazli More articles by this author , Peter BlackPeter Black More articles by this author , Martin GleaveMartin Gleave More articles by this author , and Alan SoAlan So More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2016.02.2501AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES The treatment options to prevent recurrence and progression in patients with non-muscle invasive bladder cancer (NMIBC) are limited. Heat Shock Protein 27 (Hsp27) is a cytoprotective protein that has been linked to therapeutic resistance and disease progression in many malignancies, including bladder cancer (BCa). OGX-427 is a 2nd generation (2′-MOE) antisense oligonucleotide that potently inhibits Hsp27 expression in vitro and in vivo. A phase I clinical study was performed to assess the safety, pharmacodynamic (pd) and biologic effects of intravesical treatments of OGX-427 using a presurgical dose-escalating design for patients with BCa. METHODS Eligible patients include patients with Ta, T1 or carcinoma-in-situ and candidates for transurethral resection of the bladder tumour (TURBT), or patients with muscle invasive BCa (>cT2) and candidates for radical cystectomy (RC). Patients were treated with intravesical OGX-427 on days 1, 3, 5 and 8 and then underwent surgery on day 9-12. Dose was escalated after tolerability and safety assessment for each cohort at 20uM, 50 uM, 100uM, 250uM, 500uM, and 750uM. Dose limiting toxicities (DLTs) and adverse events (AEs) were assessed. Pharmacodynamic properties of OGX-427 was determined through assessment of knockdown of Hsp27 by immunohistochemistry of tumours after treatment. Anti-tumour activity was measured by response to treatment 9-12 days after OGX-427 intravesical administration by a secondary TURBT for patients with NMIBC and RC patients with muscle invasive disease compared to baseline histology. RESULTS Twenty-four patients were enrolled. 23 patients had NMIBC and one had cT2 disease treated by TURBT and RC, respectively. OGX-427 decreased expression of Hsp27 in tumours assessed after treatment when compared to pre-treatment. Pathological staging of surgical specimens revealed that eight patients had initial complete responses (p0). The remaining NMIBC patients had pTa tumours that ranged from G1-G2; the sole patient undergoing RC had pT2aG3 bladder cancer. No significant drug-related adverse events were reported; no dose limiting toxicity was observed CONCLUSIONS OGX-427 was well tolerated intravesically with minimal toxicity. Our results indicate early evidence for activity of OGX-427 treatment in NIMBC that requires confirmation in phase II/III studies. © 2016FiguresReferencesRelatedDetails Volume 195Issue 4SApril 2016Page: e142 Advertisement Copyright & Permissions© 2016MetricsAuthor Information Sebastian Frees More articles by this author Eliana Beraldi More articles by this author Kim Chi More articles by this author Ladan Fazli More articles by this author Peter Black More articles by this author Martin Gleave More articles by this author Alan So More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Antisense inhibition of apolipoprotein (a) to lower plasma lipoprotein (a) levels in humans

Epidemiological, genetic association, and Mendelian randomization studies have provided strong evidence that lipoprotein (a) [Lp(a)] is an independent causal risk factor for CVD, including myocardial infarction, stroke, peripheral arterial disease, and calcific aortic valve stenosis. Lp(a) levels >50 mg/dl are highly prevalent (20% of the general population) and are overrepresented in patients with CVD and aortic stenosis. These data support the notion that Lp(a) should be a target of therapy for CVD event reduction and to reduce progression of aortic stenosis. However, effective therapies to specifically reduce plasma Lp(a) levels are lacking. Recent animal and human studies have shown that Lp(a) can be specifically targeted with second generation antisense oligonucleotides (ASOs) that inhibit apo(a) mRNA translation. In apo(a) transgenic mice, an apo(a) ASO reduced plasma apo(a)/Lp(a) levels and their associated oxidized phospholipid (OxPL) levels by 86 and 93%, respectively. In cynomolgus monkeys, a second generation apo(a) ASO, ISIS-APO(a)Rx, significantly reduced hepatic apo(a) mRNA expression and plasma Lp(a) levels by >80%. Finally, in a phase I study in normal volunteers, ISIS-APO(a)Rx ASO reduced Lp(a) levels and their associated OxPL levels up to 89 and 93%, respectively, with minimal effects on other lipoproteins. ISIS-APO(a)Rx represents the first specific and potent drug in clinical development to lower Lp(a) levels and may be beneficial in reducing CVD events and progression of calcific aortic valve stenosis.