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Report

Teaser, summary, work performed and final results

Periodic Reporting for period 1 - ClickGene (Click Chemistry for Future Gene Therapies to Benefit Citizens, Researchers and Industry)

Teaser

Summary

ClickGene pursues two main objectives:
1. We provide world-class training to early stage researchers (ESRs) in the areas of synthetic click chemistry, nanotechnology, liposomal drug delivery and nucleic acid (DNA and RNA) chemistry,
2. We generate scientific excellence and technological innovation in the areas of gene silencing and epigenetic base detection by generating:
2.1. Unique gene silencing tools that interact with DNA in a fundamentally different way compared with current start-of-art-technology on the market,
2.2. New liposomal and nanoparticle drug delivery agents, and
2.3. Novel fluorogenic probes for epigenetic base detection in high-throughput PCR assays.

These goals are important for society as this network will train the next generation of leading scientists in critical research fields that are required to maintain and enhance EU competitiveness in the areas of gene editing technology, nanotechnology, and nucleic acid chemistry. As such, this network will enhance biopharmaceutical, biodiagnostic, and agricultural EU societal sectors. Secondly, the scientific and technological advantages for developing new tools for gene editing, epigenetic base detection, and nanoparticle drug delivery are all important for mankind and strive toward improving healthcare and the availability of cutting-edge biomedical diagnostics for European citizens and beyond.

Work performed

The ClickGene project began in January 2015 and has recruited 14 ESRs across 9 beneficiary host organisations. Our main research focus to-date lies within gene silencing and epigenetic base detection and these activates, so far, have resulted in the publication of 15 peer reviewed scientific publications, many of which have appeared in premier journals (e.g. Angewandte Chemie [I.F. = 11.709], Nucleic Acids Research [I.F. = 9.202] and Chemical Communications [I.F. = 6.567]). ClickGene researchers have also contributed scientific talks and poster presentations to numerous scientific conferences throughout Europe. For example, our network has interfaced with COST Action CM1201 â€˜Biomimetic Radical Chemistryâ€™ and together we have held two joint research meetings (Dublin, July 2015 and Grenoble, April 2016) that allowed ClickGene ESRs to present (and engage) their research to the wider European scientific community. As part of our Dissemination and Exploitation plan a range of activates have occurred over the previous 24 monthsâ€”these include the development of a dedicated website (www.clickgene.eu), the establishment of a Twitter account (@ClickGene_H2020), and the promotion of nucleic acid click chemistry through a range of workshops and information sessions tailored toward high school students and teachers. From 16 deliverables planned for the reporting period, 12 are completed and 4 are delayed but are on track. Furthermore, all milestones for the reporting period have been achieved.

Final results

Scientific progress, beyond the state-of-the-art, is occurring within the ClickGene network. Our three goals for scientific and technological advancement involve:

1. Developing unique gene silencing tools. We have made significant inroads within this field and demonstrated that small molecule DNA oxidants, alone, can be applied for protein engineering [Kellett at al., Chem. Commum., 2015, 51 and Kellett et al., Synlett., 2015, 26], and as potential chemotherapeutic agents [Kellett et al., ACS Chem. Biol., 2016, 11]. Our current focus is to produce target-specific gene knockout agents. This goal is quite advanced with ESRs now establishing methodologies for click chemistry reactions to alkyne modified nucleic acids. We also aim to enzymatically incorporate DNA oxidants within target oligos with a basis for this technology recently established by the Hocek group [Hocek et al., ACS Chem. Biol., 2016, 11 and Hocek et al., Angew. Chem. 2016, 128]. Should we succeed in our goal to establish this technology, the ClickGene project will reach a major landmark in the design and development of new gene silencing tools.

2. Liposomal and nanoparticle drug delivery agents. ESRs have engineered exciting new nanoparticle drug delivery agents that respond to external stimuli (e.g. heat and pH). Nano-carrier sizes can be further controlled to allow delivery of specific cargo; this factor will prove important for the delivery of new gene silencing agents and epigenetic probes being developed in other WPs. Liposomal transfection agents are also under investigation here [Ferreri et al., PLoS One, 2015, 11 and Chatgilialoglu et al., Diagnostics, 2016, 7], and, in collaboration with our industrial beneficiary LipiNutaGen Srl, the design of specific liposomal carriers that can selectively target human cancer cells is underway. Experimental methods in the development of new transfection technology are also underway by the Kellett group [Kellett et al., Nucleic Acids Res., 2017, 45].

3. Probes for epigenetic base detection. The focus in this area is the development of gene targeting probes to localize epigenetic marks and specific sequences. The probe strands are constructed using click chemistry. In a first step new DNA probes were generated by the Carell group in combination with quantitative PCR to detect single epigenetic fdC (formyl-deoxycytidine) bases at defined positions in the genome [Carell et al., Angew. Chem. 2016, 55]. This WP also aims to develop an epigenetic detection system using highly parallel array assay development. To this end, the modification of epigenetic detection system developed by the Carell group is underway so that detection can be achieved using ultrasensitive mass spectrometry; if this can be achieved it could allow the parallel monitoring of hundreds of fC positions in a short period, which would revolutionise epigenetic monitoring. Two other important avenues for probe development are being pursued by baseclick GmbH where click-chemistry based assay development for (trans)-gene detection is underway, along with work at ATDBio Ltd. where high-throughput and large-scale production of clickable fluorogenic probes based on triplex forming oligonucleotides (TFO) are under development to identify epigenetic bases in target duplex strands.