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Modelling Neuron-Glia Networks into a drug discovery platform for Pain Efficacious Treatments - Sofia ref.: 116072

Periodic Reporting for period 3 - NGN-PET (Modelling Neuron-Glia Networks into a drug discovery platform for Pain Efficacious Treatments - Sofia ref.: 116072)

Reporting period: 2019-04-01 to 2020-03-31

Chronic pain affects approximately 1.5 billion people worldwide. Currently, there are 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 overall objectives of NGN-PET were: 1) to understand which mechanisms are most important in clinically common neuropathic conditions and 2) to develop efficient assays that capture the key mechanisms that can be used for compound screening.
The project has accomplished with the tasks planned. We have identified nociceptor induced gene expression in purified nociceptive sensory neurons (SN), both in naïve and injured states in mice and rats. Some of the identified targets were evaluated in the in vivo models and in vitro assays. Proteome profiling was performed on samples obtained from naïve and vincristine-treated (VCR) animals, identifying potential biomarkers for disease prevention and diagnosis for neuropathic pain, and new targets for drug development. The two animal models of neuropathic pain used in the project were set up and pharmacological studies performed with reference compounds, tool compounds for new targets and combinations of both. Spinal microdialysis in awake rats was validated with reference compounds. Macrophage medium transfer experiments and co-culture experiments point to a complex, bi-directional crosstalk between glial cells and neurons. Our co-culture systems have been evaluated under different sensitization options, measuring the effects of various mediators on nociceptor sensitization. Additional experiments were performed on myeloid cells and were incorporated into a publication (Liang et al. Pain, in press). 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 SN. Commercially available neonatal rDRGs and explanted primary rDRGs from naïve and VCR rats cultivated in miniaturized format showed a good morphology and the functional presence of pain relevant channels. Phenotypic analysis of neurons in 384-well format was proved and first results using a VCR were obtained. In relation to hiPSC work, a cell line was registered and used throughout the project. The protocols for the production, cryopreservation, transport and differentiation of SN, hiPSC-derived macrophage (iPSCdMΦ), and co-cultures have been developed. We established the lineage-specific TRPV1-mCherry reporter line, that proved the nociceptive sensory identity, and the iPSCdMΦ differentiation and definition of co-culture conditions, that maintains the full macrophage and SN functionality. SN differentiation was also implemented at Axxam, demonstrating robustness of the method and its applicability for larger screening purposes. We have demonstrated the feasibility to perform a FLIPR-based, HTS functional assay, using co-cultures or monoculture of hiPSC-derived SNs and supernatant from macrophages activated with LPS. This assay has been used for the study perturbations in the neuro-excitability induced by Veratridine. On the other hand, we developed a HCS phenotypic assay in 384-well plate format, to monitor modification in the neurite structure and outgrowth, using VCR as control of neurotoxicity. The assay can be used for in vitro functional and phenotypic testing and screening of compounds.
Exploitation: the SMEs, AXXAM and L&B, and the research institute NMI, have already included the assays and models used and developed in NGN-PET as new services and are being offered to the scientific and academic partners. At KCL, several cell-type specific RNA-seq datasets have been generated and published. A public database has been created to allow other biologists easy access. EST and GRT, have already analyzed two targets, but the list of genes and proteins identified is huge and the validation of new targets is a continuous process at both pharmaceutical companies. Those targets with sufficient efficacy, pharmacokinetic features, and safety profile, could become the starting point of a program to find a new modality for pharmacological intervention. GRT and EST have plans to incorporate the developed co-cultures to their HCS/HTS or MEA systems in the project workflows. The two neuropathic pain models have been already used for other internal projects at both companies. The hiPSC-derived cell types will be part of the drug discovery process from now on. Besides the work done with macrophages, several other glial cells have to be developed and studied, one by one or in combination. The exploitation plans will contribute to support European leadership in achieving a sounder knowledge on chronic pain pathways, crucial in guiding the discovery of effective analgesics and of clinically useful biomarkers and help improve the health care of patients.
Dissemination: the partners have actively worked to disseminate the project results by participating to scientific conferences, social media, publications of project proceedings and results, targeting both the general public and the research community.
The main ambition of the project was to understand which mechanisms are most important in clinically common neuropathic conditions, and consequently, developing efficient assays that capture the key processes that can be used for compound screening. In this respect, the project has produced several relevant results. Examples of concrete results obtained that are of a significant are:
- new knowledge, advancement in target identification and markers, new predictive models for the discovery of effective therapies. Knowledge has been made accessible by oral and poster congress communication, partners websites and publications. A transcriptomic database, NIPPY - Neuro-Immune interactions in the Periphery, generated by KCL is available at - http://rna-seq-browser.herokuapp.com/
- a number of lab- and industrial-scale assays for drug identification and validation compatible with patch clamp, MEA, High-Content and Hight-Throughout Screening platforms, based on primary rodent DRG cells and iPSC-derived sensory neurons, glial and immune cells. Assays and related cell lines are accessible for use by scientific and industrial community through the collaboration with NGN-PET partners (AXXAM – www.axxam.com , L&B - www.lifeandbrain.com , NMI – www.nmi.de)
The scientific objectives were ambitious and the timings tight. The consortium managed the difficulties generated with diligence and commitment. Defined risks and some unforeseen ones have been addressed reaching agreements on the mitigation plan to apply. The use of the scientific results and the novel screening systems will foster the generation of IP, 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.