Posters and Guidelines
Thank you for considering to present your work as a poster at Oligo 2023 Oxford. Please submit your poster abstract online within the advertised deadlines.
- Page size: Prepare your poster as you would normally do for printing. You can prepare your poster in sizes A1 or A0, but the page size of your poster is not important as the posters will be presented digitally. However, if you are attending in-person and would like to bring along a physical poster, prepare your poster in A1 portrait format (59cm wide x 84cm long). Do not laminate your poster, or use heavy printing material.
- Naming your poster files: Name your poster files as follows: <your surname>-Oligo23-Poster.pdf | <your surname>-Oligo23-Poster.png | <your surname>-Oligo23-Poster.jpg, etc. For example, for David Jones, name your file as Jones-Oligo23-Poster.pdf. DO NOT name your poster files as, e.g., Oxford-poster, poster2021v, Oxford-oligo-poster. Such files will be automatically rejected.
- Poster submission: Submit your final poster as both PDF and JPG/PNG files via the link below no later than 22nd March 2023. Late posters may not be included in the symposium programme. Please DO NOT send your poster or abstract files by email. Please ensure you send us the very final version of your poster (as well as your poster abstract), as once published, it cannot be replaced.
Poster presentation: Posters will be made available via a secure page to the conference participants before the meeting. There will be two ways to interact with the poster presenters:
- the participants will be able to ask questions via the Zoom chatbox during the mid-conference breaks; and/or
- the participants can post their questions on Twitter at any time using the meeting hashtag #OligoOx23, as well as the poster specific hashtag (given under each poster abstract) – do tag @LPMHealthcare in your tweets.
Any further information about the poster presentations at this digital meeting will be available in the future.
Before uploading your poster, you must make sure that you follow ALL of the instructions above!
(Presenters in Bold)
Accepted poster abstracts (Unedited) will be published below. 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 emailing OligoOxford@gmail.com.
Clicked Modified Antisense Oligonucleotides Targeting Hutchinson–Gilford Progeria Syndrome (HGPS)
Asmaa E Abdelrahman1,2, Alejandro G Molina3, Wiktoria M Hanszke3, Poul Nielsen3, Eva A Christensen1
1Department of Green Technology, Faculty of Engineering, University of Southern Denmark, Odense, Denmark
2Department of Photochemistry, National Research Centre, Dokki, Giza, Egypt
3Department of Physics, Chemistry and Pharmacy, University of Southern Denmark, 5230 Odense M, Denmark
Hutchinson-Gilford Progeria Syndrome (progeria) is a rare progressive genetic disease which occurs due to a heterozygous single-point mutation in the LAMNA gene. The mutation activates an alternative splice site and consequently, a truncated version of lamin A (progerin) is formed, which produces wobbly lobulated nuclei and rapid cell apoptosis and swollen nuclei.Earlier, we reported two treating pathways using well-designed ASOs to stop the translation of the progerin via blocking the mRNA, or sterically hindering the spliceosome to bind to the pre-mRNA of the progerin and form the mRNA. Also, it was reported that phenyl-triazole-2´-OMe uridine showed promising results in inducing exon skipping in vitro due to increasing the affinity of the RNA.Herein we carried out repetitive treatments of progeroid cell cultures with phenyl-triazole uridine modified ASOs which are engineered to prevent the ribosome in the cytoplasm from translating the mRNA of the progerin. Whereas four chemically synthesized monomers were incorporated into ASOs, aiming for higher stability and better binding to the mRNA of the progerin. Phenyl-triazole analogues were attached via click chemistry to the 5-position either of uridine or 2´-OMe-uridine. Six modified ASOs were transfected every other day for ten days in triplicates. Confocal microscopy was used to image the DAPI-stained fixed cells. The change in the nuclear morphology was analysed using Nikon NIS-Elements; 400 to 500 nuclei were analysed per condition. Phenyl triazole-2´-OMe uridine-modified ASO provides an enhancement in the nuclear morphology of the progeroid cells. Where the treated cells show regular cell growth with a similar nuclear area to healthy fibroblasts with the same passage number of cells.Further biological assays i.e., western blots, PCR, proliferation, and toxicity are still running to analyse the gene expression at protein and RNA levels.
Chemical synthesis of 4’-modified nucleoside analogues
Caecilie M M Benckendorff, Mieke Guinan1, Mark Smith2, Gavin J Miller1
1School of Chemical and Physical Sciences and Centre for Glycoscience Research, Keele University, Keele, Staffordshire, ST5 5BG, United Kingdom
2Riboscience LLC, 428 Oakmead Pkwy, Sunnyvale, CA 94085, USA
Oligonucleotide therapeutics have emerged as a state-of-the-art drug modality, paving the way for the chemotherapeutic intervention of hard-to-treat diseases such as viral infections, cancers and genetic diseases. Comprised of short chains of synthetic single or double stranded DNA or RNA, they exhibit their pharmaceutical potential with high specificity and in a multitude of mechanisms throughout the different phases of pathogenesis, depending on their structure. Non-modified oligonucleotide sequences tend to have poor pharmacokinetic properties, and as such, the inclusion of chemically modified nucleotide units has been instrumental to the development of successful oligonucleotide therapeutics with improved metabolic stability, cellular uptake and target binding affinity. Modifications such as phosphorothioate linkages have shown to improve stability towards enzymatic cleavage as well as decrease hydrophilicity thereby improving cellular uptake. Likewise, the inclusion of modified sugar moieties can enhance metabolic stability and may induce a conformational change that favours target binding. Within the context of sugar modifications, we have developed scalable synthetic access to 4’-thionucleosides, alongside carbocyclic nucleoside analogues wherein the furanosyl oxygen is replaced CHF and CF2 groups, further enriching the library of building blocks available for the synthesis of novel therapeutic oligonucleotide candidates.
Towards precision targeting of non-coding RNAs using oligonucleotide catalysts
Maria J Donde, Adam M Rochussen, Saksham Kapoor and Alexander I Taylor
Cambridge Institute of Therapeutic Immunology & Infectious Disease (CITIID), Jeffrey Cheah Biomedical Centre, University of Cambridge, Cambridge, UK
In principle, RNA-cleaving oligonucleotide catalysts (ribozymes and DNAzymes) could modulate the expression of virtually any gene of interest with high specificity and activity independent from cellular machinery, reducing off-target effects compared with other approaches. However, clinical applications have been hampered by the limitations of DNA & RNA: most examples require non-physiological conditions (e.g. pH >7, ≥10mM [Mg2+]) for efficient activity and are susceptible to digestion by nucleases. To tackle these issues, we have recently engineered artificial oligonucleotide enzymes (XNAzymes) fully composed of 2’-fluoroarabino nucleic acid (FANA) that retain activity under physiological conditions. We have shown they are capable of allele-specific knockdown of mRNA inside cells, measured by RT-ddPCR and sequencing of cleavage products, as well as SARS-CoV-2 genomic RNA, measured by inhibition of authentic viral replication. As a proof of concept, we also examine the potential for XNAzymes to target individual members of disease-associated non-coding RNA families (microRNAs in the miR17-92 cluster, miR-21 or the hY5 ncRNA). We find that in vitro bespoke FANA catalysts specifically or preferentially cleave their cognate targets and outperform analogous RNA or DNA catalysts under quasi-physiological conditions.
High-throughput selection and characterisation of aptamers on optical sequencers
Alissa Drees, Christian Ahlers, Markus Fischer
Hamburg School of Food Science, Institute of Food Chemistry, University of Hamburg, Germanyalissa.email@example.com High-Throughput Sequencing-Fluorescent Ligand Interaction Profiling (HiTS-FLIP) is the first experiment to enable the quantitative measurement of millions of DNA-protein interactions in parallel. For this, it utilizes the capability of optical sequencers to perform fluorescence-based assays on the immobilized DNA-clusters subsequent to sequencing. Using different protocols between sequencing and screening of binding, HiTS-FLIP can be used to study a wide range of interactions of fluorescent molecules with ssDNA, dsDNA, RNA and even peptides. We successfully modified the common sequencer “Illumina MiSeq” to automatically perform custom high-throughput binding screenings. Only minor hardware modifications, i.e. additional tubing and an additional external valve, were required, making HiTS-FLIP for the first time available to anyone with access to a MiSeq. To demonstrate the potential of HiTS-FLIP, we exploited its’ capacity to enable high-throughput selection as well as characterisation of high-affinity aptamers (single-stranded oligonucleotides that can form defined three-dimensional structures by intramolecular interactions) for different medical-relevant target proteins within a period of a few days. Compared to the conventional selection of aptamers by Systematic Evolution of Ligands by Exponential Enrichment (SELEX), the required effort was significantly reduced while the information content about the binding interactions was drastically increased, providing potential data for machine learning approaches.
Steps toward on-chip electrochemical detection of microRNA biomarkers from liquid biopsies for early detection of prostate cancer
Xiaotong Meng, Loukia Petrou, Ahmad Kennan, Daanyaal Khan, Danny O’Hare* and Sylvain Ladame
Department of Bioengineering, Imperial College London, 86 Wood Lane, London, W12 0BZ, UK
Over 1 in 8 men will be diagnosed with prostate cancer in their lifetime in the UK causing more than 12,000 deaths each year. However, this predicament can be changed if prostate cancer is diagnosed at an early stage when more easily treatable and before it has started to metastasise. Traditional methods for prostate cancer diagnosis include prostate-specific antigen (PSA) blood test and digital rectal examination (DRE). Whilst DRE is highly invasive and depends strongly on doctor’s clinical experience, PSA lacks specificity and was shown to lead to overdiagnosis and overtreatment. MicroRNAs (miRNAs) in bodily fluids are an emerging class of minimally invasive molecular biomarkers for early diagnosis of a broad range of diseases, including prostate cancer. However, to be implemented in clinic, new diagnostic tools based on miRNA detection require technology that are both highly sensitive (due to the low natural abundance of miRNA in bodily fluids) and highly sequence-specific (due to the high sequence homology between miRNAs). Here I will present our work toward the development of a novel electrochemical miRNA sensor by using bespoke peptide nucleic acid (PNA) probes directed against miR-141, a miRNA overexpressed in the blood of prostate cancer patients. Two PNA probes was designed and synthesised for on-chip capturing and sensing miR-141. A biotinylated PNA capture probe was immobilized on streptavidin-coated screen-printed carbon electrode. The electrochemical detection was then carried out via a redox-labelled PNA sensing probe used as an electrochemical reporter. We will be reporting here the design, engineering, and in vitro study of our PNA-based sensor.
Rational design of a minor groove directed Dinuclear Zinc(II) Complex for targeted gene therapy
Georgia Menounou1, Reabetswe Zwane1, Leila Tabrizi1, Andrea Exrleben2, Andrew Kellett1
1SSPC, the Science Foundation Ireland Research Centre for Pharmaceuticals, School of Chemical Sciences, Dublin City University, Glasnevin, Dublin 9, Ireland
2School of Chemistry, National University of Ireland, Galway, Ireland.
The development of metal-based therapeutics is a research field of broad interest that can lead to effective treatment against various forms of cancer. Several polynuclear metallodrug candidates containing endogenous transition metals, have displayed excellent cooperative interactions at the drug-DNA interface. Here, we report the DNA binding properties of a new dinuclear Zn2+ metallodrug containing a poly-pyridyl caging scaffold, [2,7-Zn2(tetra-(2-pyridyl)-NMe-naphthalene)Cl4] (2,7-Zn2TPNap). Zn2TPNap is a high-affinity DNA binder with apparent binding constant (Kapp) in the order of 107 M(bp)−1. High-throughput fluorescence quenching assays with synthetic copolymers poly[d(A-T)2] and poly[d(G-C)2] revealed the minor groove as the preferential binding site and thermal melting studies showed selective destabilization of poly[d(A-T)2]. Viscosity analysis on ctDNA displayed a significant intercalating (hydrodynamic) binding effect, similar to that observed for ethidium bromide. Studies involving supercoiled pUC19 DNA reveal condensation and topoisomerase I inhibition. To probe drug-induced conformational changes, a series of circular dichroism experiments were conducted. With ctDNA, Zn2TPNap perturbed the secondary DNA structure with an associated loss of helicity. Structural distortions of poly[d(A-T)2] arose, while in poly[d(G-C)2] the β-N glycosidic and H-bonds were altered. To investigate the sequence-specific binding mode of Zn2TPNap a selection of palindromic dodecamers were examined with varying sequence context. Here, concentration-dependent exposure to the palindromic sequences suggests a binding preference of the metallodrug to TATA-rich sequences as indicated by helical unwinding and alterations in the π-π stacking spectral region. Finally, Pre-associative molecular docking studies with specific oligomers support selective minor groove intercalation.
Intracellular trafficking of bioreducible poly(amidoamine) nanoparticles for mRNA delivery
Adriano P Pontes1, Saketh R Ranamalla2, Steffen van der Wal1, Karin Roelofs1, Ioan Tomuta2, Laura B Creemers3, Jaap Rip1
120Med Therapeutics B.V., Galileiweg 8, 2333 BD Leiden, The Netherlands
2Department of Pharmaceutical Technology and Biopharmacy, University of Medicine and Pharmacy “Iuliu Hațieganu”, 41 V. Babes Street, 400012 Cluj-Napoca, Romania
3Department of Orthopedics, University Medical Center Utrecht, Heidelberglaan 100, 3584 CX Utrecht, The Netherlands
Disulphide bond-poly(amidoamine) (PAA) is a cationic and bioreducible polymer, with potential use as a nanocarrier for mRNA delivery in the treatment of several diseases including osteoarthritis (OA). Successful transfection of joint cells with PAA nanoparticles (NPs) was shown previously but cell uptake and endosomal escape were not studied in detail. In addition, cationic nanoparticles are often associated with off-target uptake by tissue-resident macrophages, which also triggers inflammation-induced nanoparticle toxicity. Coating the nanoparticles with polyethylene glycol (PEG) can overcome these toxic effects and improve colloidal stability. In this study, C28/I2 human chondrocytes were transfected with PEG-coated PAA NPs loaded with EGFP mRNA for confocal imaging of intracellular trafficking and evaluation of transfection efficiency. This system showed promising features as a gene delivery vehicle, such as small particle size (about 60 nm), excellent monodispersity and electro-neutral surface. Uptake was higher for cationic (uncoated) PAA NPs, although virtually all cells showed internalization of PEG-coated NPs. Furthermore, endosomal leakage/escape over time was shown in live C28/I2 cells with the fluorescent probes calcein and LysoTrackerTM. Finally, we applied a D-optimal experimental design to test different mRNA-to-polymer ratios and dosages, thus obtaining an optimal formulation (42.5 w/w polymer to mRNA ratio and 640 ng mRNA per well) with up to ≈80% of EGFP-expressing cells and without toxic effects. Together, the biocompatibility and high transfection efficiency of this system is a promising tool for intra-articular delivery of therapeutical molecules in OA treatment.
New RNA modifications for translation studies
Tania Sanchez-Quirante1,2, Katarzyna Grab3, Joanna Kowalska3, Michal Hocek1,2
1Institute of Organic Chemistry and Biochemistry Czech Academy of Sciences. Flemingovo nam.2, 16610 Prague 6, Czech Republic
2Department of Organic Chemistry, Faculty of Science, Charles University in Prague, Hlavova 8, CZ-12843 Prague 2, Czech Republic
3Division of Biophysics, Institute of Experimental Physics, Faculty of Physics, University of Warsaw, Pasteura 5, 02-093 Warsaw, Poland
The study of RNA has increased considerably in the recent years. It is known that some modifications in the RNA have biological effects, as increased stability and thus having consequences in translation efficiency. Previously in our lab it was studied the efficiency of the T7 polymerase to incorporate different modifications. The result of those studies reported that this polymerase is able to incorporate small modifications such as methyl and ethyl substituents. For translation it is also important the role of the 5’ cap, we chose m7GpppAmpG, a trinucleotide cap that it is known for having a high capping efficiency. In this work, a library of 8 nucleoside triphosphates (NTPs), have been synthetized and have been incorporated during in-vitro transcription (IVT) by the T7 polymerase. Different length templates have been used to synthetize short RNAs such as 35nt and 70nt long, with different amount of NTPs incorporated. On the other hand, mRNA was synthetized using Pjet-Gaussia luciferase as a DNA template. So far, in-vitro translation studies were performed using Rabbit Reticulocyte System (RRS) and evaluating the translation efficiency with the luminescence activity of Pjet-Gaussia luciferase.
The effect of N6 -methyladenosine modifications on mRNA stability and expression in CHO cells
Or Skornik, Niall Barron
National Institute for Bioprocessing Research and Training (NIBRT), Dublin, Ireland
Chinese hamster ovary (CHO) cells are the most frequently applied host cell system in the pharmaceutical industry. Current CHO processes can typically yield 3-10 g/L of product, exceeding productivity of many other cell lines. These yields have come about via optimized expression systems as well as genetic engineering advances including overexpression and silencing of specific genes. RNA-based technology is now recognised as a promising approach for cell engineering applications, with the advantages of a fast translation, no size limitation of the transcript delivered into the cell, higher control over the expression of proteins and an easier and cheaper manufacturing process. RNA has not been a popular tool for cell engineering applications until recently due to its immunogenicity. However, recent ground-breaking studies have shown that mRNA post-translational modifications pose a solution to this problem, furthermore, RNA post translational modifications contribute to the transcript stability and expression level. N6 -methyladenosine (m6A) modification is the most abundant mRNA modification and has been proven to improve the mRNA transcript’s properties.In this work, we aimed to transfect CHO cells with modified mRNA transcripts and investigate the modification’s influence on the transcript when different concentrations of modified bases were incorporated randomly into the transcript.Modified mRNA transcripts for green fluorescent protein (GFP) were created using in vitro transcription (IVT) reaction supplemented with 6 different concentrations of m6A bases and adenosine bases (100%, 50%, 25%, 12.5%, 6.25%, 0%). m6A presence in the transcripts was evaluated by Dot Blot. Transcripts were then transfected into CHO cells. We evaluated the GFP expression levels in the transfected CHO cells using flow cytometry.We show that m6A modified RNA bases, when implemented in an arbitrary manner throughout the mRNA transcript, had a negative effect on the transcript’s expression levels. Our results show a negative correlation between the concentration of m6A modified bases used in the IVT reaction and the expression level of the mRNA transcript, as the lowest concentration of modified bases used in the reaction resulted in the highest level of GFP expression, which gradually decreased as mRNA transcripts containing higher concentrations of modified bases were used.Overall, this study shows the negative effects of randomly incorporated m6A modified bases on mRNA expression levels, suggesting that this modification is closely controlled in vivo and is able to support mRNA stability and expression levels only when incorporated in specific transcript locations.
Third generation sequencing of epigenetic DNA
Beth Searle1, Andrew Kellett1, Markus Müller2, Thomas Carell2
1School of Chemical Sciences, Dublin City University Glasnevin, Dublin 9, Dublin, Ireland
2Department of Chemistry, Ludwig-Maximilians Universität München, Butenandtstr, 5–13,81377, Munich, Germany.
The discovery of epigenetic bases has revolutionised our understanding of disease and development. Meanwhile, advances in sequencing technology have presented the opportunity to sequence an increasing number of genomes in greater detail. ‘Third generation’ platforms enable the generation of sequencing reads multikilobases in length, facilitating coverage of regions of the genome considered inaccessible via the shorter read lengths produced using ‘next generation’ technology. In order to understand genetic function with single nucleobase precision, it is imperative methodology is developed to sequence the full spectrum of epigenetic DNA modifications. Reviewed are techniques at the intersection of sequencing technology advances and epigenetic sequencing chemistry: chemical and enzymatic methodologies developed and adapted for sequencing modified cytosines on Oxford Nanopore and PacBio’s SMRT devices. These developments provide insight into potential future developments in chemistries targeted towards epigenetic signatures.
Trans-cyclooctene- and bicyclononyne-linked nucleotides for click modification of DNA with fluorogenic tetrazines and live cell metabolic labelling and imaging
Ambra Spampinato1,2, Tomáš Kraus1, Milan Vrábel1, Erika Kužmová1, Veronika Sýkorová1 and Michal Hocek1,2
1Institute of Organic Chemistry and Biochemistry, Czech Academy of Sciences, Flemingovo nam. 2, CZ-16610 Prague 6, Czech Republic;
2Dept of Organic Chemistry, Faculty of Science, Charles University, Hlavova 8, Czech Republic
2- or 4- trans-cyclooctene (TCO) or bycyclononyne (BCN) were chosen as potential reactive groups for fluorogenic IEDDA click labeling with tetrazines as tool for bionalytical applications. Several examples of click chemistry TCO-bearing antibodies were used for tumor pre-targeting, cellular imaging etc. A portfolio of 2`-deoxycitidine triphosphates linked to TCO or BCN through shorter propargylcarbammate or longer triethylene-based spacer were synthetised and used as good substrates for KODXL DNA polymerase to incorporate in modified oligonucleotides. TCO- and BCN- modified triphosphates were delivered to live cells using a synthetic transporter SNTT1 and subsequentely treated with several fluorogenic coumarin-based tetrazines. In particular we have found that PEG3-linked TCO and BCN modified triphosphates were efficiently incorporated into genomic DNA and well reacted with coumarine-tetrazines for imaging of DNA synthesis in cellulo. For flow citometry the combination of BCN-linked nucleotide with TAMRA-linked tetrazine was also efficient for staining of DNA. In comparison with previous methodologies, this method, for in cellulo metabolic labelling and imaging of DNA synthesis, is advantageous in term of timing and semplicity. Acknowledgment: The work was funded by Czech Science Foundation (20-00885X).
Suppression of SARS-CoV-2 Replication with Stabilized and Click-Chemistry modified siRNAs
Annika J Tölke1, Franziska R Traube1, Marcel Stern2, Sabine Schneider1, Markus Müller1, Oliver T Keppler2, Thomas Carell1
1Department of Chemistry, LMU Munich, Butenandtstr. 5-13, 81377 Munich, Germany
2Max von Pettenkofer Institute and Gene Center, LMU Munich, Feodor-Lynen-Str. 25, 81377 Munich, Germany
The emergence of more transmissible or aggressive variants of SARS-CoV-2 requires the development of antiviral medication that is quickly adjustable to evolving viral escape mutations. Here we report the synthesis of chemically stabilized small interfering RNA (siRNA) against SARS-CoV-2. The siRNA can be further modified with receptor ligands such as peptides using Cu(I)-catalysed click-chemistry. We demonstrate that optimized siRNAs can reduce viral loads and virus-induced cytotoxicity by up to five orders of magnitude in cell lines challenged with SARS-CoV-2. Furthermore, we show that an ACE2-binding peptide-conjugated siRNA is able to reduce virus replication and virus-induced apoptosis in 3D mucociliary lung microtissues. The adjustment of the siRNA sequence allows a rapid adaptation of their antiviral activity against different variants of concern. The ability to conjugate the siRNA via click-chemistry to receptor ligands facilitates the construction of targeted siRNAs for a flexible antiviral defence strategy.