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Report

Teaser, summary, work performed and final results

Periodic Reporting for period 2 - NGN-PET (Modelling Neuron-Glia Networks into a drug discovery platform for Pain Efficacious Treatments - Sofia ref.: 116072)

Teaser

Summary

Chronic pain is a complex disease process affecting approximately 1.5 billion people worldwide. Currently, there are very few effective treatments available, a high number of non-responder patients, and the side-effects highly reduce treatment compliance. Beside neurons, the role of glial cells in the development and maintenance of chronic pain is currently widely accepted. There is considerable potential for developing novel analgesic drugs that target neuronal-glial interactions.
The development of new screening tools promises a great variety of benefits starting from reducing timings and cost for launching new treatments with better efficacy and less side-effects, reducing the economic and social cost of chronic pain.
The overall objectives of the project are:
1. To understand which mechanisms are most important in clinically common neuropathic conditions, and whether these explain the high susceptibility of women to develop such symptoms.
2. To develop efficient assays that capture the key mechanisms that can be used for compound screening.

Work performed

The NGN-PET project has accomplished with the tasks planned until the end of the second year.
In WP2, we identified nociceptor induced gene expression in purified nociceptive sensory neurons, both in a naÃ¯ve and injured state (D2.3). Targets from this list will be selected to be evaluated in the in vivo models and in vitro assays. The two animal models of neuropathic pain (spinal nerve ligation and chemotherapy-induced neuropathy) were set up (D2.2), and a dose-response curve for reference compounds obtained. Spinal microdialysis in awake rats was validated with reference compounds. The Certificates/Personal licenses (D2.1) were delivered in due time.
Macrophage medium transfer experiments and co-culture experiments performed in WP3 point to a complex, bi-directional crosstalk between this cell type and neurons. Our co-culture systems are in place, and different sensitization options have been evaluated, measuring the effects of various mediators on nociceptor sensitization (D3.1; D3.2).
Neuron purification experiments in combination with MEA and calcium imaging experiments successfully identified a functional role for non-neuronal cells in establishing / promoting excitability of sensory neurons (D3.3).
We demonstrated the feasibility to culture in miniaturized format both commercially available neonatal rDRGs and explanted primary rDRGs from naÃ¯ve and vincristine-treated rats. Cells cultivated in this format for about one week showed a good morphology and the functional presence of pain relevant channels such as TRPV1. The suitability of 384-well plate format for phenotypic analysis of physiological relevant model of neuronal cells was proved. We have shown the first results supporting the effect of compounds inducing pain, such as chemotherapeutic drugs.
WP4 started tissue collection from naÃ¯ve and vincristine-treated animals. These samples are being used for the proteome profiling in this WP and for in vitro assays in WP3.
WP5 registered at the â€œhuman pluripotent stem cell registryâ€ (hiPSCreg) the hiPSC line used within the project (D5.1). The protocol for the production and cryopreservation of early stage SN has been successfully developed (D5.2), they have been functionaly characterised (D5.3), and the protocols for differentiation improved (D5.5). We have also generated constructs, and genetically modified hIPSC lines, harboring reporter systems for lineage-specific markers (D5.4). Protocols for the differentiation of UKBi013-A iPSC into glial-like cells, for co-culture development, were implemented and validated. Generation of constructs for monitoring calcium flux in hIPSC derived nociceptors is completed. We have also established the differentiation procedure of relevant glia cells for co-cultures (D5.6). Finally, cell culture systems to study SN neuron-glia interaction in stimulated vs. non stimulated conditions and identification of human specific (pain) signatures has been set up and will be further characterized.
In WP6, using naÃ¯ve hiPSC-derived sensory neurons, we have started working in setting up assays, in semi-automated manner, in the miniaturized 384 well-plate format, based on fluorescent dyes and/or antibodies suitable for HTS or HCS platforms. Different conditions, in terms of coatings, cell densities, media, timepoints and fluorescent dyes have been used in order to identify the best performing protocol, in terms of signal to background, pharmacology and reproducibility of the results.
It was demonstrated the feasibility to introduce semi-manual steps of automation in miniaturized format, for the use of hiPSC-derived sensory neurons in HTS and HCS platforms.
In WP1, all project management tools and procedures are running. At time of reporting all actions planned for internal communication and dissemination for the second year of project were undertaken at the due delivery date. Two meetings of the overall consortium were held: one in Bresso, at Axxam and the second in Bonn at Life & Brain. Due

Final results

The main ambition of NGN-PET is to understand which mechanisms are most important in clinically common neuropathic conditions to develop efficient assays that capture the key processes that can be used for compound screening. The project has already produced key results for advancing towards those specific objectives. In particular, transcriptomics data were generated in purified neurons from pain models, data pointing to the bi-directional cross-talk between macrophages and neurons were generated and hiPSC genetically modified reporter cell lines were generated.
Transcriptomic data will be used to identify targets for therapeutic intervention and identify biomarkers. We will use also these results for set up in vitro cell cultures of hiPSC-derived sensory neurons and glial cells, and to adapt said hiPSC-derived cultures to high-throughput drug screening systems.
The use of these novel systems will foster the generation of intellectual property, new chemical entities suitable for drug development, products and services, increasing the competitiveness of European SMEs and pharmaceutical companies, with the ultimate objective of identifying novel curative therapies for neuropathic pain.
Exploitable results are planned to be obtained at the end of the project: M36, D1.5 will be delivered, outlining the strategy for exploiting the project outcomes. This report will also lay out the different options for creating a new framework that allows expanding the current project activities beyond the project life.
These results will also contribute to support European leadership in achieving a sounder knowledge on pathways, crucial in guiding the discovery of effective analgesics and of clinically useful biomarkers and help improve the health care of millions of patients.