Posters and guidelines
Thank you for considering to present your work as a poster at Oligo 2021 Oxford Virtual.
Digital poster submission deadline: Prepare your poster as you would normally do for printing, and submit your final poster as both PDF and JPG/PNG files via the link below no later than 16th April 2021. Late posters may not be included in the conference programme. Please DO NOT send your poster files by email.
Naming your poster files: Name your poster files as follows: <your surname>-Oligo21V-Poster.pdf | <your surname>-Oligo21V-Poster.png | <your surname>-Oligo21V-Poster.jpg, etc. For example, for David Jones, name your file as Jones-Oligo21V-Poster.pdf. DO NOT name your poster files as, e.g., Oxford-poster, poster2021v, Oxford-oligo-poster. Such files will be automatically rejected.
Poster presentation: Posters will be made available via a secure page to the symposium 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 #OligoOx21V, as well as the poster specific hashtag (given under each poster abstract) – do tag @LPMHealthcare in your tweets.
The poster presenters should regularly check Twitter for any questions about their posters before, during and after the meeting, and post their answers on Twitter using appropriate hashtags, as above.
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.
Synthesis of a vancomycin-modified uridine to be included in aptamers
#OligoOx21V, #CFigazzolo
Chiara Figazzolo1,2,3, Fabienne Levi-Acobas1, Marcel Hollenstein1
1Institut Pasteur, department of Structural Biology and Chemistry, Laboratory for Bioorganic Chemistry of Nucleic Acids, CNRS UMR 3523, 28 rue du Docteur Roux, 75015 Paris, France.
2Université de Paris, 12 rue de l’École de Médecine, 75006 Paris, France.
3Centre de Recherches Interdisciplinaires CRI, 8 Rue du Charles V, 75004 Paris, France
Antibiotic resistance (ABR) is one of the biggest public health challenges of our time and occurs when bacteria develop mechanisms that protect them from the effects of antibiotics. In this work, we explore the first steps towards combatting ABR by attaching antibiotics on the scaffolds of aptamers. The aim of this work was to synthesize a novel deoxyuridine nucleotide bearing a vancomycin moiety and assess its capability to be incorporated into DNA via enzymatic synthesis, and hence proving its potential application in SELEX experiments. Vancomycin’s resistance has been widely observed in S. aureus which can alter a peptidoglycan terminus, resulting in reduced vancomycin binding and failure to prevent bacterial wall synthesis. The identification of a vancomycin-bearing aptamer would potentially use the aptamer binding properties to mechanically force the vancomycin in the binding site and allow its function despite the presence of the aforementioned mutation. Initially, a suitably protected alkyne-modified deoxy-uridine was synthesized, triphosphorylated and then reacted with a vancomycin modified with an azide group. Subsequently, a biochemical characterization was performed to assess the substrate acceptance of the nucleotide by polymerases. The PCR showed that the nucleotide is accepted by the polymerases Deep Vent and Vent exo–. The primer extension (PEX) demonstrated the formation of full-length products with the polymerases Phusion, Q5, Therminator, Deep Vent and Vent exo–. PEX also showed the possibility to incorporate a series of vancomycin modified nucleotides in a row. Finally, we demonstrated that a template containing a vancomycin-modified uridine can be converted into unmodified DNA without mutations or misincorporations which is an important prerequisite for SELEX experiments. In conclusion, this work proves the possibility to enzymatically synthesize oligonucleotides containing a vancomycin-modified uridine and use them in SELEX experiments.
New lipid-oligonucleotide conjugates containing long-chain sulfonyl phosphoramidate groups
#OligoOx21V, #AAFokina
Alesya A Fokina1,2, Alina S Derzhalova1,3, Oleg A Markov3, Yasuo Shiohama4, Timofei S Zatsepin5,6, Masayuki Fujii7, Marina A Zenkova3, Dmitry A Stetsenko1,2
1Department of Physics, Novosibirsk State University, 2 Pirogov Str, Novosibirsk 630090, Russia
2Institute of Cytology and Genetics, Siberian Branch of the Russian Academy of Sciences, 10 Lavrentiev Ave, Novosibirsk 630090, Russia
3Institute of Chemical Biology and Fundamental Medicine SB RAS, 8 Lavrentiev Ave, Novosibirsk 630090, Russia
4Center of Molecular Biosciences, University of the Ryukyus, 1 Senbaru, Nishihara, Okinawa 903-0213, Japan
5Center of Life Sciences, Skolkovo Institute of Science and Technology, Innovation Centre Skolkovo, Bld 1, Bolshoi Bulvar, Moscow region 121205, Russia
6Chemistry Department, Lomonosov Moscow State University, Bld 3, 1 Leninskie Gory, Moscow 119991, Russia
7Department of Biological and Environmental Chemistry, Kindai University, 11-6 Kayanomori, Iizuka, Fukuoka 820-8555, Japan
Novel DNA and RNA lipid conjugates with one to four [(4-dodecylphenyl)sulfonyl]phosphoramidate or (hexadecylsulfonyl)phosphoramidate group at the internucleotidic position on the 3’- or 5’-end were obtained by automated solid-phase synthesis using Staudinger reaction between the phosphite triester and the respective sulfonyl azide. Oligodeoxynucleotide conjugates with one or two hydrophobic groups were found to be non-cytotoxic for mouse or human macrophages, and able to enter the cells in a carrier-free manner. Lipid-conjugated siRNAs targeting repulsive guidance molecule a (RGMa) have shown a comparable gene silencing activity in PK-59 cells to unmodified control siRNA when delivered by Lipofectamine-mediated transfection.
Tyrosine-modified PEI for highly efficacious and biocompatible siRNA delivery in vitro, ex vivo and in vivo
#OligoOx21V, #MKarimov
Michael Karimov, Alexander Ewe, Achim Aigner
Rudolf-Boehm-Institute for Pharmacology and Toxicology (Clinical Pharmacology), University of Leipzig, Medical Faculty, Härtelstraße 16-18, 04107 Leipzig, Germany
The delivery of small interfering RNAs (siRNA) is a promising approach for the target-specific knockdown of pathologically overexpressed genes. A major challenge is the safe and efficient delivery of siRNAs esp. for in vivo applications. The cationic polymer polyethylenimine (PEI) is a promising delivery system. However, while their high charge densities efficiently complex and protect the payload, PEIs are also associated with high cytotoxicity, colloidal aggregation and often moderate transfection efficacy. Chemical modifications are a promising strategy for further improvement. We developed a set of novel tyrosine-modified PEIs. These were thoroughly studied for optimal siRNA complexation conditions and other physicochemical properties. The biological and toxicological behaviour of the siRNA complexes was characterized in vitro. Finally, the most promising complexes were further evaluated ex vivo and in vivo. Gene silencing efficacies up to >90% were found in different cell lines and not associated with appreciable cytotoxicity. Incubation in the presence of serum, freezing or lyophilisation of nanoparticles indicated excellent colloidal and biophysical stability. In ex vivo studies based on a tumor tissue slice model, a profound knockdown of GAPDH was found on mRNA and protein levels. In a pre-clinical in vivo study in colon carcinoma xenograft-bearing mice, tyrosine-PEI/siRNA nanoparticles targeting the anti-apoptotic oncogenes survivin and PLK-1 showed strong tumor-inhibitory effects and knockdown of the target proteins by ~50% without adverse effects. In conclusion, the chemical modification of PEI with the amino acid L-tyrosine strongly improved the physicochemical stability, reduced the toxicity and increased the bioactivity of nanoparticles. Thus, we present tyrosine-modified PEIs as particularly efficient and biocompatible platform for siRNA delivery, as demonstrated in vitro, ex vivo and in different tumor models in vivo.
A new method for oligonucleotide conjugation by amide bond formation on solid phase at a modified phosphate group after Staudinger reaction
#OligoOx21V, #KKlabenkova
Kristina V. Klabenkova1,2, Ekaterina A. Burakova1,2, Sergei N. Bizyaev1,3, Alesya A. Fokina1,2, Dmitry A. Stetsenko1,2
1Department of Physics, Novosibirsk State University, 2 Pirogov Str., Novosibirsk, Russia
2Institute of Cytology and Genetics, Siberian Branch of the Russian Academy of Sciences, 10 Lavrentiev Ave., Novosibirsk, Russia
3N.N. Vorozhtsov Novosibirsk Institute of Organic Chemistry, Siberian Branch of the Russian Academy of Sciences, 9 Lavrentiev Ave., Novosibirsk, Russia
Synthetic oligonucleotide derivatives that can modulate RNA metabolism by e.g., antisense or RNAi mechanism, gained widespread interest as promising therapeutic agents. The most widely used are the DNA and RNA analogues with modified phosphate group such as phosphorothioates due to facile chemical synthesis and favourable biological properties. However, most oligonucleotide derivatives are difficult to deliver into target cells and tissues, which complicates their clinical development. A potential solution to this problem can be oligonucleotide conjugation with molecules that improve cellular uptake and biodistribution, such as polyamines, which introduce positive charges into oligonucleotides.The Staudinger reaction on solid phase between an organic azide and internucleotidic phosphite is a convenient method for chemical modification of oligonucleotides. In this work, pentafluorophenyl and 4-nitrophenyl esters of 4-carboxybenzenesulfonyl azide were used to introduce an activated carboxyl group to the 5′-terminal internucleotidic position of an oligonucleotide via Staudinger reaction. After treatment with excess primary alkyl amine and usual work-up, a series of amide-linked oligonucleotide conjugates were obtained in good yield.
Analysis of polyethylenimine (PEI)-based polyplexes and lipopolyplexes for pulmonary siRNA delivery in 2D- and air-liquid interface culture
#OligoOx21V, #SNoske
Sandra Noske, Alexander Ewe, Achim Aigner
Rudolf-Boehm-Institute for Pharmacology and Toxicology, Clinical Pharmacology, University of Leipzig, Germany
RNA interference (RNAi) is a highly conserved tool for gene regulation in eukaryotic organisms. It is based on the action of small interfering RNAs (siRNAs) on a target mRNA, which is cleaved and thus degraded. The downregulation of specific gene products offers new perspectives in the treatment of diseases like cancer, viral infections or genetic disorders. Due to its large surface and high vascularization, the lung is an attractive target organ for local and/or systemic application of siRNAs. A major bottleneck, however, is the delivery of active siRNA molecules to their target site. Hence, considerable research efforts have focused on the development and refinement of carriers, for overcoming extracellular and intracellular barriers. Polymeric nanoparticles, especially PEI-based ones, and their lipopolyplexes provide a promising system for gene delivery regarding to efficacy and the absence of unspecific cytotoxicity. By measuring gene knockdown, transfection efficacies were evaluated in lung cancer cell lines in 2D-cell culture and in an air-liquid interface culture system. Biological activities of nebulized (lipo)polyplexes were comparable to their non-nebulized counterparts. The ionic strength of the complexation buffer was decisive for particle size, and the positive ζ-potential of polyplexes was shielded upon lipopolyplex formation. Transfection efficacy was also observed in the air-liquid interface culture, as determined by fluorescent siRNA uptake and by reporter gene knockdown. In 2D culture, we have identified (lipo)polyplexes with high transfection efficacies and sufficient biocompatibility, even after nebulization. First results also indicate their biological activity in a more complex air-liquid interface system, but this may be affected by their interaction with pulmonary mucus. Taken together, this provides the basis for their prospective exploration as pharmacological carriers, aiming at the pulmonary application of siRNA for therapeutic use.
Affordable-SELEX: A Novel Methodology for Developing Aptamers and Acquiring 21st Century Laboratory Skills
#OligoOx21V, #GRangappa
Gautam T Rangappa 1, Gwendolyn Stovall 1,2
1Texas Institute for Discovery Education in Science, Freshman Research Initiative, University of Texas at Austin, Austin, TX 78712, USA
2Texas Institute for Discovery Education in Science, High School Research Initiative, University of Texas at Austin, Austin, TX 78712, USA
Aptamers, synthetically derived RNA or DNA molecular recognition elements, are increasing in demand for biosensors, diagnostics, and therapeutic technologies. Aptamer selection processes oftentimes require specialized equipment and training which causes them to be restrictive due to their high-cost burden. To counter the aforementioned issue, the Aptamer Lab of the Freshman Research Initiative at the University of Texas at Austin has altered traditional SELEX processes to create a SELEX methodology that is more affordable, easy to execute, and able to be performed in parallel. Researchers performing aptamer selections with the “Affordable-SELEX” method could discover novel aptamers that have the potential to advance modern scientific applications. Currently, I am leading a team of researchers writing a manuscript detailing our adapted SELEX method for publication in the Journal of Visualized Experiments (JoVE). By marketing our research lab’s “Affordable-SELEX” method, our intent is to provide individuals interested in aptamer research an easily accessible way to begin rounds of selection. In the wide body of aptamer literature, many publications that are available do not provide a comprehensive protocol on how to perform aptamer selections. Through outlining our distinct bead-based and filter-based protocol utilizing video and written formats, we hope to provide interested researchers with a cost-efficient way to execute aptamer selections at large scale. Although the proposed protocol requires more effort to customize selection conditions compared to other methods, researchers are able to foster essential research techniques through this unique learning experience.
Evaluation of DMD transcripts after golodirsen treatment of MyoD-converted fibroblasts from 4053-101 clinical trial patients
#OligoOx21V, #RRossi
Rachele Rossi*1, 2, Marc Moore*1, 2, Silvia Torelli1, 2, Pierpaolo Ala1,2, Francesco Catapano1,2, 3, Rahul Phadke3, Jennifer Morgan1,2, Jyoti Malhotra4, Francesco Muntoni1,2.
1The Dubowitz Neuromuscular Centre, UCL Great Ormond Street Institute of Child Health, London, UK
2National Institute for Health Research, Great Ormond Street Institute of Child Health Biomedical Research Centre, University College London, London, UK
3Dubowitz Neuromuscular Centre, Division of Neuropathology, UCL Queen Square Institute of Neurology, Queen Square, London, UK
4Sarepta Therapeutics, Cambridge, MA, USA
* The authors have equally contributed to the work
Duchenne muscular dystrophy (DMD) is an X-linked, rare, neuromuscular disease caused by mutations in the DMD gene which result in a substantial reduction or absence of dystrophin protein. The commonest type of mutation is deletion of one or more exons, followed by large duplication; together they occur in 75% of DMD cases. Antisense oligonucleotides modulate exon incorporation, masking the splicing enhancer sequences or splice junctions in the pre-mRNA. This induces exon skipping, resulting in the restoration of the mRNA reading frame and translation of internally shortened dystrophin protein. Several antisense oligonucleotides are now approved, including the phosphorodiamidate morpholino oligomers (PMOs) eteplirsen and golodirsen, respectively targeting exons 51 and 53.In Study 4053-101, we demonstrated exon 53 skipping and protein restoration in all muscle biopsies from golodirsen-treated patients. There was some inter-patient variability in dystrophin protein restoration, likely related to the mechanism of PMO uptake in muscle fibres, and potentially to other factors. Here, we assess the specific exon 53 PMO-induced skipping in fibroblasts, derived from the patients enrolled in study 4053-101, that were differentiated into myotubes by lentivirally-mediated MyoD and treated with golodirsen. Single-system qPCR in treated/ non-treated patients and healthy controls revealed a high exon skipping rate in all post-treatment cells. Next, we evaluated the transcript 5’-3’ imbalance and the relative amount of the restored transcript by custom FluiDMD cards. To understand the intracellular dynamics and subcellular localization of the skipped products, we used specific BaseScope probes. Finally, we verified abundance and molecular weight of the restored protein by WES. Comparison of these in vitro findings to the previous in vivo patient data may reveal why the same AON treatment has different outcomes among patients. This work was funded by Sarepta Therapeutics, Inc.
Antisense oligonucleotide-mediated knockdown of IGF-binding proteins to increase IGF-1 signalling
#OligoOx21V, #AYavas
Alper Yavas1,3, Maaike van Putten1,3, Erik H Niks2,3, Annemieke Aartsma-Rus1,3
1Department of Human Genetics, Leiden University Medical Center, Leiden, Netherlands
2Department of Neurology, Leiden University Medical Center, Leiden, Netherlands
3Duchenne Center Netherlands, Utrecht, Netherlands
Duchenne muscular dystrophy (DMD) is an X-linked neuromuscular disorder caused by the absence of dystrophin protein. There is an imbalance between muscle damage and repair due to the impaired regenerative capacity of the muscle. Insulin-like growth factor 1 (IGF-1) plays an essential role in muscle maintenance. Other than liver, IGF-1 is also expressed in skeletal muscle. Both in circulation and extracellular matrix (ECM), IGF-1 is bound to binding proteins (IGFBPs). The majority of IGF-1 is bound by IGFBP3 while other IGFBPs are also found in the circulation and ECM. These binding proteins are thought to act by inhibiting the availability of IGF-1 to its receptor, which initiates most of the actions of IGF-1. Therefore, decreasing IGFBP levels would enhance IGF-1 signaling, making it a potential therapy for the treatment of secondary defects in DMD. This can be achieved by antisense oligonucleotides (AONs) that skip out of frame exon(s), thus preventing the expression of functional proteins. In this study, two different AONs were designed to target exon 2 of both Igfbp1 and Igfbp3. The AONs were tested and optimized in C2C12 cells using lipofectamine-mediated transfection with different concentrations. To confirm exon skipping, total RNA was analyzed at 24 h post transfection and RT-PCR was performed. At least one AON was found to be effective for each gene. With these candidate AONs, transfection was repeated to assess the effect of exon skipping on protein level as well. After 48 h, proteins were isolated and downregulation of binding proteins and its effect on the downstream proteins, which are involved in IGF-1 signaling cascade, were confirmed by Western blot. Using exon skipping mediated knockdown of IGFBP1 and IGFBP3, we confirmed in vitro that IGF-1 signaling can be enhanced, making it a potential therapy to be tested in vivo. As next step, these AONs will be used in mdx mice to see whether this treatment will improve muscle quality and function.
