Posters

Oxford Symposia - 6th Annual

OLIGO 2020 OXFORD:

Antisense & Therapeutic Nucleic Acids

30-31 March, St Hilda's College, Oxford, UK

Twitter: @LPMHealthcare, #OligoOx20

Posters and guidelines

Guidelines for poster preparation

Please prepare your poster in A1 portrait format (59cm wide x 84cm long). Do not laminate your poster, or use heavy printing material. Further information about poster sizes can be found on the following link:

http://tinyurl.com/y7bf

Posters larger than A1, or those in landscape orientation, will only be displayed subject to the availability of space.

Maximum capacity 10 A1 potrait posters

Please ensure you have appropriate permissions for the publication of your abstract from the original copyright holders. Should you wish your abstract not to be published, please notify us in writing at the time of abstract submission.

>>Where can I print my poster in Oxford?

Posters will be displayed for the full duration of the symposium.

Accepted posters

(Presenters in Bold)

If your abstract has been accepted for presentation but it does not appear in the list below, please let us know as soon as possible by email on Oligomer@LPMHealthcare.com.


Using attenuated anthrax toxin to mediate the cytosolic delivery of a peptide designed to inhibit androgen-dependent mitogenic signaling in prostate cancer

Hadeer K.S. Abdelrahman, Nourhan A.M. Mahmoud, Benedita K.L. Feron and Simon C.W. Richardson*

Exogenix Laboratory, School of Science, University of Greenwich, Central Avenue, Chatham Maritime, Kent ME4 4TB, UK

Prostate cancer is the second leading cause of cancer-related death in men. Previous studies have documented androgen and sarcoma (Src) receptor activation as a key contributor to the development of prostate cancer. Based on these findings, a peptide (IP-1) was developed to inhibit the association between sarcoma and androgen receptors. The purpose of this study was to assess the impact of intracellular compartmentalization upon IP-1 bioavailability. To this end, cytosolic delivery technology derived from attenuated anthrax toxin was used. This consisted of Bacillus anthracis protective antigen (PA) of 83 KDa (PA83) and the N-terminal 255 amino acids of Bacillus anthracis lethal factor (LFn). To mediate delivery, IP-1 was fused-in-frame with LFn (LFn-IP1) and co-incubated with PA83. HeLa cells were used to evaluate the impact of the delivery system upon the cytosolic distribution of LFn-IP1. Cells were treated with 50 µg/ml PA83 and 50µg/ml Texas Red (TxR)-labelled LFn-IP1. Following incubation periods of 1, 4 and 24h, LFn-IP1-TxR appeared in the peri-nuclear region; after which the protein was documented diffusing across the cytosol overtime. Cytosolic translocation of the complex was shown to be dependent upon PA83 by repeating the previous live cell imaging experiment without PA83. Under these conditions, LFn-IP1 was imaged within perinuclear endocytic LAMP1-positive vesicles but did not translocate to the cytosol. These data demonstrate for the first time that PA can mediate the delivery of IP1-TxR into the cytosol through the PA pore. In conclusion, these data indicate the potential utility of a cytosolic delivery system to improve upon protein therapeutic bioavailability.


Pioneering siRNA-mediated protection of mammalian cells against Zika virus (MR-766) infection

Benedita K L Feron1, Joachim J Bugert2 and Simon C W Richardson1

Exogenix laboratory, University of Greenwich, School of Science, Kent, UK

Institut für Mikrobiologie der Bundeswehr, Munich, Germany

Here we present empirical data documenting the siRNA-mediated protection of cells after Zika virus (ZIKV) MR-766 infection. These siRNA sequences were designed to target well-conserved sequences across the ZIKV genome. Several delivery technologies were utilised. After the electroporation of 100nM siRNA into human hepatocyte-derived carcinoma (Huh7) cells, the Feron Zv-2 sequence (specific to the ZIKV NS3 gene) yielded a cell viability of 150.3±7.4% (SEM: n=4) (p=0.0004) relative to cells treated only with virus (33.9±12%, SEM: n=4). Further, 100nM siRNA Feron Zv-4 (specific to ZIKV 3’UTR) resulted in 119.1±11.2% cell viability (SEM: n=4) relative to the control cells treated with ZIKV (p=0.0021). Cells were electroporated with siRNA prior to ZIKV infection and viability was monitored 4 days after this. Additionally, two novel siRNA delivery systems were tested. The first utilized recombinant Bacillus anthracis PA83 (octomer forming mutants) co-incubated with the N-terminal 255 amino acids of B.anthracis lethal factor (LFn) fused-in-frame with the RNA binding domain for human protein kinase R (LFn-PKR) at a concentration of 50µg/ml (each). Here, baby hamster kidney (BHK) cells treated with 100nM siRNA Feron Zv-1 yielded 79.0±4.0% viability relative to the control (50.2±1.7%, SEM: n=3), 3 days after exposure to ZIKV (p=0.0096). Finally, HeLa exosomes loaded with siRNA Feron-Zv2 were incubated with Huh7 cells prior to ZIKV infection. For the siRNA-exosome treated cells, a viability of 123±46% (SEM: n=18) relative to 8±16% (SEM: n=18) for the same concentration of control HeLa exosomes was recorded (p=0.0416). In each instance, 0.3 MoI was used and cell viability monitored using the PierceTM Firefly Luciferase Glow Assay Kit by Thermo ScientificTM.  Here, it has been shown for the first time that siRNA can significantly reduce ZIKV-derived cell kill. Future work will require quantitating ZIKV mRNA in relation to siRNA treatment as well as testing the siRNAs and delivery systems within more complex models.


Peptidyl-Oligonucleotide conjugates to target miRNAs associated with poor prognosis in Cancer

Thomas Heyman1, David Clarke1, Olga Patutina2, Marina Zenkova2, Harmesh Aojula1 and Elena V. Bichenkova1 

1 School of Health Sciences, Faculty of Biology, Medicine and Health, University of Manchester, Oxford Road, Manchester, M13 9PT, UK

2 Institute of Chemical Biology and Fundamental Medicine SB RAS, 8 Lavrentiev Avenue, 630090, Novosibirsk, Russia

Cancer is one of the largest causes of mortality in the western world. Despite large interdisciplinary effort and considerable funding to support the development of more successful and less toxic anti-cancer therapies, surgery remains the single most effective modality for the treatment of cancer. Conventionally, chemotherapies aim to target DNA and cause enough DNA damage to induce cell death, whereas targeted therapies aim to inhibit specific proteins associated with disease. The main issues associated with these modalities are poor selectivity and off target effects resulting in severe systemic toxicity. We are currently developing novel therapeutics which recognise and cleave highly oncogenic miRNAs (i.e. miR-17, miR-21 and miR-155) that are commonly overexpressed in almost every cancer type and have associations with poor prognosis. These Peptidyl-Oligonucleotide conjugates are comprised of a recognition oligonucleotide motif attached to a catalytic peptide with cleavage capabilities. As each miRNA are posttranscriptional regulators of multiple gene targets, these therapeutics have the potential to downregulate expression of multiple genes whilst only interacting with a single type of miRNA. These novel peptidyl-oligonucleotide conjugates are fully characterised with NMR and mass spectrometry, and their binding and cleavage efficacy are assessed using various assays. The best candidates will be selected for functional, in vitro and in vivo assays. The selectivity and tuneability of this new class of drug enables them to potentially be used in countless medical applications, simply by changing the oligonucleotide recognition sequence to another target. This new type of therapeutic interventions has the potential to revolutionise the way we treat cancer and will have a positive impact on the health outcomes for patients.


Sequence-Specific Detection of Unlabelled Nucleic Acid Biomarkers Using a “One-Pot” 3D Molecular Sensor

Andrew Irwin1, Sameen Yousaf1, Alberto Saiani2, David J. Clarke1, Harmesh S. Aojula1, Aline F. Miller2, Elena V. Bichenkova1

1 School of Health Sciences, University of Manchester, Oxford Road, Manchester M13 9PT, UK

2 Manchester Institute of Biotechnology, 131 Princess Street, Manchester M1 7DN, UK

MicroRNAs are ssRNA molecules ~22 nucleotides in length, which act as post-transcriptional regulators of gene expression. They are attractive biomarker candidates with the potential to diagnose diseases at an earlier stage. However, translation into Point-Of-Care environments has been limited due to high costs, low sensitivity, poor detection limits and a lack of reproducibility across platforms. A hydrogel environment allows for greater analyte storage than a 2D chip-based design, increasing sensitivity and lowering the limit of detection. This project is focused on designing a reporter group functionalised hydrogel, capable of specifically detecting a target sequence. This work has shown sequence-specific detection of unlabelled nucleic acid biomarkers using a molecular beacon-based approach within a hydrogel environment. Thiol-maleimide coupling was used to conjugate a thiol-functionalised oligonucleotide sequence containing a fluorescein and dabcyl quencher, to a maleimide-linked peptide. Complementary ionic peptides, which self-assemble to form β-sheets, were utilised as a matrix to attach the peptide-oligonucleotide conjugate. Hybridisation between the nucleotide sequence within the molecular beacon and a complementary target sequence facilitated a conformational change, thus leading to a disruption of FRET and release of a fluorescent signal. This system was capable of recognising a “Perfect Match” (PM) target from within a longer sequence and the background signal was less than in solution. The system was also capable of detecting the PM within an oligo mixture containing various short genomic sequences, as well as discriminating between the PM and sequences containing single, double or triple residue mutations. Development of this system will involve optimising the design for effective quenching and signal generation, assessing functionality within a microfluidic environment and selection of specific miRNAs significantly increased in diseased states.


Attenuated anthrax toxin mediated siRNA cytosolic translocation requires intraluminal vesicles

Nourhan A. M. Mahmoud, Hadeer K. S. Abdelrahman, Benedita K. L. Feron and Simon C. W. Richardson*

Exogenix Laboratory, School of Science, University of Greenwich, Central Avenue, Chatham Maritime, Kent, UK

Post-endocytic intracellular trafficking is an established rate limit for the effective use of membrane non-permeant drugs such as plasmids, gene editing proteins, small-interfering RNA (siRNA) and antisense oligonucleotides (ASOs). Of the many proposed solutions to this problem, we have focused on the use of attenuated, recombinant Bacillus anthracis toxin (ATxn) which, when fused-in-frame to an RNA binding protein domain can enhance the activity of siRNA and ASOs by ~300%, without any measurable toxicity. Wildtype ATxn has been shown to utilize intraluminal vesicles (ILV) for the efficient cytosolic delivery of lethal factor (LF). Here, it was hypothesized that specific inhibitors of ILV formation or function, would reduce the cytosolic translocation efficiency of the ATxn-mediated delivery of siRNA (via the formation of a PA83 : LF truncation (LFn) fused to either the RNA binding domain for protein kinase R (LFnPKR)) or a protein, i.e. diphtheria toxin A chain (LFnDTA). The possibility that wheat germ agglutinin (WGA) may inhibit ILV formation reducing cytosolic translocation efficiency was considered, due to the protein’s ability to cross-link cell membrane. WGA’s ability to reduce cytosolic access was tested by treating HeLa cells with 50 µg/ml PA83 and 10 µg/ml LFnDTA and monitoring cell viability recorded as 23.0±0.5 % (n=3±SEM). After the addition of 5 µg/ml WGA prior to PA83 and LFnDTA cell viability recorded as being 112.9±0.3% (n=3±SEM). Having established that WGA could inhibit PA translocase activity, WGAs effect on siRNA translocation was evaluated by monitoring siRNA activity in HEK293 (SC008) cells overexpressing beta-galactosidase. In this instance, gene knockdown was achieved using 100nM siRNA, 50 µg/ml PA83 and 50 µg/ml LFn-PKR and measured 48h after transfection. This resulted in 11.5 ± 0.3% target gene expression (n=3±SEM). Optimal inhibition of PA::LFnPKR::siRNA translocation by WGA was documented at 3µg/ml, resulting in 48.2±2.1% target gene expression (n=3±SEM), revealing significant reduction in siRNA activity (p=0.008). These findings support the hypothesis that WGA inhibits ILV formation and that ILV formation is required for the efficient PA83-mediated translocation of both LFnDTA and LFnPKR::siRNA into the cytosol via ILVs. This would indicate that PA is not simply destabilizing the limiting membrane of the endosome in order to facilitate cytosolic access.


Exclusivity in nucleic acid targeting – Preclinical validation

Petra Mazancova1,2, Veronika Nemethova1,2, Andrea Babelova3,4, Alena Gabelova3, Michal Selc3,4, Kristina Kopecka3, Boglarka Nemethova1,2, Lucia Uhelska1,2, Alexandra Poturnayova5, Angelika Batorova1,6, Antonia Hatalova6, Filip Razga*1,2

1 Faculty of Medicine Comenius University, Bratislava, Slovak Republic

2 Selecta Biotech SE, Bratislava, Slovak Republic

3 Cancer Research Institute, Biomedical Research Center SAS, Bratislava, Slovak Republic

4 Centre for Advanced Material Application SAS, Bratislava, Slovak Republic

5 Institute of Molecular Physiology and Genetics, Centre of Biosciences SAS, Bratislava, Slovak Republic

6 Clinic of Hematology and Blood Transfusion, University Hospital, Faculty of Medicine, Medical School   Comenius University, Slovak Medical University, Bratislava, Slovak Republic

Although delivering authentic innovations today is more complex than ever, we anticipated to develop a reliable and precise technology with a great potential to resolve the promiscuity of antisense oligonucleotides and facilitate a global reform in the design and hence applicability of antisense therapeutics. Our findings provided early evidence that our prominent technology provides exclusive discrimination between direct native homologues and target fusion sequences that could not be achieved by today’s approaches. Basic cytotoxicity studies showed no toxicity of the antisense compound at the concentrations tested, even of those exceeding the biologically effective dose ≥20-fold. Functional in vitro validation on the protein level showed a decrease in target fusion protein to 0 % by day 5 upon treatment. The antisense oligonucleotide moreover exhibited spontaneous delivery (100 million molecules per cell) into hard-to-transfect cells including suspension ones, which is an extraordinary feature given the global problem of achieving efficient uptake of therapeutic oligonucleotides. Unprecedented exclusivity towards leukemic cells was evidenced by selectively induced cell death of target positive cells (versus 94% viability of target negative cells at day 6 under the same experimental conditions) in a dose-dependent manner. The unmatched accuracy of the presented proprietary antisense platform and the vision of a facile technology that can completely spare healthy cells opens the prospect that, after many years of biology-driven research, an alternative treatment option for cancer patients might be on the horizon. This work was supported by the Slovak Research and Development Agency under Contract No. APVV-15-0215 and APVV-16-0579, and by VEGA Grant No. 2/0094/15. Filip Razga received support within the SASPRO Programme (Project No. 0057/01/02) co-funded by the European Union and the Slovak Academy of Sciences.


Aptamers – promising diagnostic tools in leukemia

Andrea Bábelová1,2, Katarína Macková3, Kristína Kopecká1, Michal Šelc1,2, Veronika Némethová4,5, Filip Rázga4,5, Alexandra Poturnayová3

1 Cancer Research Institute, Biomedical Research Center SAS, Bratislava, Slovakia

2 Centre for Advanced Material Application, Slovak Academy of Sciences, Bratislava, Slovakia

3 Institute of Molecular Physiology and Genetics, Centre of Biosciences SAS, Bratislava, Slovakia4 Faculty of Medicine, Comenius University, Bratislava, Slovakia 5 Selecta Biotech SE, Bratislava, Slovakia

Leukemia is a hematological disorder arising from bone marrow (BM), characterized by abnormal proliferation of BM cells, contributing to series of life-threatening symptoms. Leukemia immunotherapy has been dominant via using synthetic antibodies to target cluster of differentiation (CD) molecules, nevertheless the cytotoxicity and immunogenicity limits its development. Oligonucleotide aptamers are a class of small molecule ligands that can specifically bind to their targets on cell surfaces with high affinity. Aptamers have great potential in cancer therapy due to their unique physical, chemical, and biological properties. Especially in oncology, aptamer nanomedicine may perform better than conventional or antibody-based chemotherapeutics due to superior specificity compared to the former and improved stability compared to the latter. In this work we used DNA aptamers specific for leukemic cells. Chronic myelogenous leukemia (CML), a malignancy of pluripotent hematopoietic cells, seriously affects patient bone marrow, peripheral blood and lymphatic system. Common detection methods have a low specificity and require invasive bone marrow aspiration, which is a painful procedure for the patients. Therefore, there is room for a simple, rapid and specific diagnostic method for CML. Highly sensitive acoustic method – quartz crystal microbalance (QCM) was used for tracking the interactions between aptamer and different CML cell lines (BV-173, K562). These interactions were monitored in real time and enabled determination of binding kinetic constants between aptamers and monitored cell surface receptors. Using confocal microscopy, we investigated the localization of bound aptamers on the surface of cells. This work was supported by APVV-15-0215, APVV-16-0579, and by VEGA Grant No. 2/0094/15 and 2/0113/15. F. Rázga received support within the SASPRO Programme (Project No. 0057/01/02) co-funded by the European Union and the Slovak Academy of Sciences.


Aptamers as Reversible Sorting Ligands for Preparation of Cells in Their Native State

Martin D Requena, Bethany Powell Gray, Michael D Nichols, Bruce A Sullenger

Department of Surgery, Duke University, USA

Selective isolation of cells based on cell-surface biomarkers is an invaluable tool in many research and clinical applications, including analysis of circulating tumor cells, hematopoietic stem cell transplantations, and cancer immunotherapy. Though antibody-based magnetic-activated cell sorting (MACS) and fluorescence-activated cell sorting (FACS) are effective techniques for isolation of cells from complex mixtures on the basis of expression of a specific biomarker, antibodies cannot be easily removed from cells. We have developed a MACS- and FACS-compatible method to reversibly label and purify cells using aptamers and matched oligonucleotide antidotes. To demonstrate the utility of this method, the epidermal growth factor receptor (EGFR)-binding antagonistic aptamer E07 was immobilized on magnetic beads and used to isolate a purified population of EGFR(+) cells from a mixture of EGFR(-) and EGFR(+) cells. An antisense oligonucleotide antidote complementary to a region of E07 reverses aptamer binding thereby simultaneously detaching the cells and restoring receptor function. Quantitative western blot analysis of cell lysates after E07-FACS showed no statistically significant difference in EGFR autophosphorylation following incubation with epidermal growth factor compared to unsorted cells. We also used E07-MACS to recover labeled EGFR(+) cells from whole blood added at concentrations equivalent to 5% the white blood cell count. These results suggest aptamers and reversal antidotes can be used to isolate rare cell types in their native state for a wide variety of sensitive applications such as CAR-T cell therapy or mechanistic receptor signaling studies.


Lipidated nucleic acids (LiNAs) in oligonucleotide delivery: Liposomal delivery and bioactivity of LiNAs

Erik B. Pedersen1, Jesper Wengel1, Luigi. E. Xodo2 and Stefan Vogel1

1Nucleic Acid Center, Institute of Physics and Chemistry, University of Southern Denmark, DK-5230 Odense M, Denmark

2Department of Medical and Biological Sciences, P. le Kolbe 4, 33100 Udine, Italy

KRAS is mutated in >90% of pancreatic ductal adenocarcinomas (PDAC). As its inactivation leads to tumour regression, mutant KRAS is considered an attractive target for anticancer drugs. In this study we report a delivery strategy for a G4-decoy oligonucleotide that sequesters MAZ (myc-associated zinc-finger), a transcription factor essential for KRAS transcription. Delivery is based on the use of liposomes functionalized with lipid-modified G4-decoy oligonucleotides and a lipid-modified cell penetrating TAT peptide. The potency of the strategy in pancreatic cancer cells is demonstrated by cell cytometry, confocal microscopy, clonogenic and qRT-PCR assays. Liposomal delivery of LiNAs for I) decoy G4-quadruplexes and II) microRNA 216b derived LiNAs is presented. I) Decoy G4-quadruplexes formed by G-runs 2-3-4-5 showed the strongest affinity for MAZ which activates the transcription of KRAS. A TINA-modified quadruplex (twisted intercalated nucleic acid) showed a strong bioactivity, as it suppressed KRAS, inhibited proliferation, activated apoptosis in Panc-1 cancer cells, inhibited tumour growth. To improve the delivery we used liposomes to which we anchored noncovalently the lipid modified G4-decoy LiNA. The liposome-anchored G4-decoy efficiently internalize in pancreatic Panc-1 cancer cells where it reduces the metabolic activity, the clonogenicity as well as the level of KRAS transcript. II) miR-216b is aberrantly downregulated in pancreatic ductal adenocarcinoma (PDAC). We found that KRAS, whose mutant G12D allele drives the pathogenesis of PDAC, is a target of miR-216b. To suppress oncogenic KRAS in PDAC cells, we designed single-stranded (ss) miR-216b mimics with unlocked nucleic acid (UNA) modifications to enhance their nuclease resistance and applied a liposomal delivery strategy. We found that these versatile liposomal formulations suppressed oncogenic KRAS in PDAC cells like as reported for G4-decoy LiNAs.


Antisense oligonucleotide-mediated rescue of protein expression in fibroblasts and neurons derived from a patient with a rare neurological disorder caused by loss-of-function mutations in the TECPR2 gene

Luis A Wiliams1, Wei Tseng1, Valeriya Baru1, Hansini Upadhyay1, Christina Ambrosi1, Monika Pichler1, Amy Elder1, David Gerber1, Alan S Kopin2,3 and Graham Dempsey1

Q-State Biosciences, 179 Sidney Street, Cambridge MA 02139, USA

Luke Heller TECPR2 Foundation, USA

Molecular Pharmacology Research Center, Tufts University School of Medicine, Boston MA 02111, USA

Mutations in TECPR2 are the cause of an ultra-rare neurological disorder characterized by intellectual disability, impaired speech, motor delay and hypotonia evolving to spasticity, central sleep apnea and premature death (SPG49, OMIM # 615031). Little is known about the biological function of TECPR2, and there are currently no available disease-modifying therapies for this disease. To address this urgent medical need, we have implemented an antisense oligonucleotide (ASO) exon-skipping strategy targeting the c.1319delT (p.Leu440Argfs) mutation. To model the disease in vitro, we utilized patient-derived fibroblasts and induced pluripotent stem cell-derived (iPSC) neurons homozygous for the p.Leu440Argfs mutation that results in a premature stop codon within TECPR2 Exon 8. Both patient-derived fibroblasts and neurons showed lack of TECPR2 protein expression. We designed and screened ASOs targeting sequences across the TECPR2 Exon 8 region to identify molecules that induce Exon 8 skipping and thereby remove the premature stop signal. TECPR2 Exon 8 skipping restored expression of a TECPR2 protein variant lacking Exon 8-encoded sequence (TECPR2-DeltaEx8). Optimization of ASO sequence generated a candidate with 24 nM potency in patient-derived fibroblasts. To examine potential functional rescue induced by the candidate ASO, we used iPSC-derived neurons to analyze the neuronal localization of TECPR2-DeltaEx8 compared to full-length TECPR2. Neuronal localization of both protein forms showed clear similarities. In addition, we applied Q-State’s proprietary all-optical electrophysiology platform Optopatch to characterize the patient-derived neurons and identified synaptic activity features associated with loss of TECPR2. We are currently examining rescue of this potential functional phenotype with the ASO candidate. Ongoing studies are underway to advance this ASO therapeutic candidate through an expanded access IND for a patient with this severe, life-threatening disorder.