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Report

Teaser, summary, work performed and final results

Periodic Reporting for period 1 - PAIN-Net (Molecule-to-man pain network)

Teaser

Summary

Neuropathic pain affects 5% of the general population and 40% of patients with neurological diseases, and has a key role in the pathophysiology of cancer pain that affects up to 50% of patients in the early disease stage and 30% of survivors, causing an enormous social burden. Treatments are inadequate with less than 50% of patients achieving 50% of pain relief at best, while up to 30% of cancer pain patients experience insufficient analgesia. Signatures of individual susceptibility to pain and analgesic responsiveness are urgently needed to improve patientsâ€™ management. Such advances are expected to originate from integrated clinical, basic science and entrepreneurial research readily translating scientific findings into benefits for patients. To consolidate these aims, a new generation of scientists with wide knowledge in neuropathic pain, focused research skills and experience in the interaction with biotechnology companies is needed. The PAIN-Net programme supports such talented and inspired early stage researchers. Their research projects, embedded in an advanced molecule-to-man pain network, contribute to better understanding individual susceptibility to pain and analgesics responsiveness based on next generation sequencing, whole exome sequencing, epigenetics and pharmacogenomics studies, nociceptor and sodium channel functioning based on biophysics and proteomics studies, targeted analgesics based on high-throughput screening, targeted analgesic delivery based on encapsulated cell bioreactor implants, and to the development and extensive characterisation of the first knock-in mouse models of sodium channel-related neuropathic pain based on the CRISPR-Cas technology.

Work performed

ESR1 has included patients in the existing Propane database, developed a database for the pharmacogenomic studies, and has been trained in additional testing and investigations (neurophysiology, corneal confocal microscopy, Sudoscan, staining and counting skin biopsies).
ESR2 has enrolled 71 new patients for the MOLO study, and is working on the analysis of the database. ESR2 has also contributed to the development of a study protocol for the validation of an algorithm for the diagnosis of neuropathic cancer pain.
ESR3 is implementing the analysis of skin biopsy on different targets from patients with painful neuropathy carrying sodium channel variants, either sporadic or part of families. Other pain-related skin biopsy targets will be analysed in terms of protein expression, also in the CRIPSR-CAS mouse models.
ESR4 has performed the assessment of neuropathic patients and has been trained in neurophysiological techniques to study peripheral nerves small fibers (microneurography, Evoked Potentials). ESR 4 also performed microneurographic recordings of neuropathic pain patients and healthy volunteers.
ESR5 has been trained to execute an experimental approach (Literature review, Instruments datasheets, Matlab documentation, Videolectures, Courses, Animals handling, Advanced programming language skills, Microneurography techniques, Software development, Software Testing (Cross-checking of results with existing softwares, Blind protocols, Repetition of trials in order to reach a statistical significance, Wild-type animal models against nerve injured models)
ESR6 has finished the MIP-NGS analysis of ten VGSC genes (SCN1B-4B, SCN3A, SCN7A-11A) of 362 patients, and twenty ion channel genes of 230 painful-DPN and 644 SFN patients of 224 painful-DPN, 314 painless-DPN and 532 painful-SFN patients. Recently, ESR6 started a collaboration to test a potentially pathogenic variant in HCN1 by patch-clamp analysis, and is working on the functional testing of 5 potentially pathogenic variants ANO3 by calcium-imaging.
ESR7 has devised a workflow for the WES analysis and updated the current in house bioinformatics pipeline with new versions of the software and databases, that will be run on the PROPANE and the PAIN-NET data once available. ESR7 added new process to check for structural variations and long indels that will be studied on PROPANE (ongoing process) and PAIN-NET cohorts.
ESR8 has developed the experimental design to perform the epigenetic study on DNA methylation patterns in painful and painless small fiber neuropathy patients, with different etiology of the pathological condition (idiopathic/diabetes/cancer). ESR8 also elaborated the sample collection protocol and established contacts with recruiting partners to initiate sample collection. ESR8 applied the bioinformatic pipeline to different datasets to analyse whole-epigenome data from subjects recruited within PAIN-Net once available.
ESR9 acquired expertise in isolation and recording of DRG neurons from mice and learnt principles of patch-clamp recordings in the voltage- and current-clamp modes. ESR9 learnt data analysis for assessing biophysical properties of mutant sodium channels and for assessing firing properties of DRG neurons expressing mutanmt sodium channels. ESR9 also applied acquired expertise in acquiring and analysing data for the characterization of mutant sodium channels from patients with pain disorders.
ESR10 developed and optimized the experimental protocol that will be used on the human Nav 1.7 and 1.8 HEK293 cell lines through the automated patch-clamp (IonFlux 16 apparatus) and optimized the hERG assay protocol to investigate possible adverse effects of compounds.
ESR11 has obtained one mouse model using CrispR/Cas9. ESR12 defined the strategy to develop another model and to characterise it. ESR13 has been recruited and started working on his project. The mouse model of neuropathic chronic itch has been created.

Final results

PAIN-Net offers a training programme combining academic and industrial expertise with theoretical knowledge and complementary skills, shaping a new generation of European neuroscientists fully equipped to explore the complexity of neuropathic pain. The local training-through-research each ESR will receive while conducting her/his individual project, will be integrated with a well-designed system of secondments, network-wide events and ESRs/group interactions. This will grant a level of integration impossible to achieve in a national context, which will be further implemented by networking with neuroscience PhD courses of the Universities participating in the PAIN-Net consortium, thus fostering transfer of the PAIN-Net model into existing and/or new study plans. This will innovate the structure of neuroscience ESR training in Europe, setting the ground for training collaborations that may extend beyond the funding period of this ETN.
In parallel, PAIN-Net research activities will generate fundamental advances in the definition of individual susceptibility to pain and responsiveness to analgesics. The pathogenesis of pain in most patients is unknown and no biomarkers are available yet. Overall, not all individuals develop pain and it is currently not possible to predict who is more or less susceptible among those with similar risk exposure. Such variability remains unexplained yet. By performing research towards the identification of new therapeutic targets and strategies for pain treatment, while simultaneously training researchers to this end, the PAIN-Net project will strengthen the European innovation capacity â€“ with the aim of translating scientific findings in the complex field of pain into solutions able to benefit a larger percentage of patients - which is lagging behind.