Background & Aims
Human genomic approaches are rapidly identifying more and more genes that are significantly associated with chronic pain phenotypes. However, methods to functionally validate and prioritize novel targets are lacking. In this study, the aim was to investigate if lentiviral knock-down (KD) combined with high-capacity electrophysiology could be a viable approach to functionally validate pain targets. A proof-of-concept study was performed where neuronal excitability (key hallmark of pain) was evaluated after KD of two well-known pain targets (Nav1.7 and TrkA). After a positive outcome, the methodology was applied to screen ten novel pain targets.
Methods
Dorsal root ganglia neurons (DRGs) from adult rats were cultured in a 384-well format. Cells were transduced with lentiviral shRNA against target or scrambled control at 1 day in vitro (DIV) and neuronal excitability and cell health was evaluated at 9 DIV. At the day of the functional experiments, electric field stimulation (EFS) was applied to the DRGs while recording the response on an optical electrophysiology platform (Deng et al., 2023; Sidders et al., 2018). Strictly standardized mean differences (SSMD) were calculated as a measure of effect size and quality control (QC). KD was confirmed for Nav1.7 and TrkA on gene-expression level and cell health was evaluated by high content imaging with markers for cell nuclei and the neuronal network (?-III-tubulin).
Results
Lentiviral shRNA targeting NaV1.7 resulted in a significant decrease in excitability for three out of four oligos tested. Treatment with oligo B resulted in the strongest effect on excitability, with an SSMD >2. Targeting TrkA resulted in a significant decrease in excitability for two oligos. Treatment with oligo A resulted in the strongest effect, with an SSMD >1. With the assumption that NaV1.7 is a fairly strong control and TrkA a moderate control, an SSMD of ?2 and ?1 respectively would be accepted as a QC criterium in a screening setting. For selected active oligos, the KD was confirmed on gene expression level.
Subsequently, a screen of 10 pain targets was performed. With the QC criterion that SSMD should be ?2 for NaV1.7 KD, 73 % of the 42 tested plates successfully passed QC. Of the 10 tested targets, 5 targets were modulating neuronal excitability after lentiviral KD with an effect size of at least 20% in two out of three test occasions and without affecting cell health.
Conclusions
In conclusion, we have validated an approach to assess the effect of lentiviral KD on excitability in peripheral neurons as well as identified two suitable positive controls. Moreover, we have demonstrated the utility of the approach to identify targets that modulate neuronal excitability.
References
Deng, L., Dourado, M., Reese, R. M., Huang, K., Shields, S. D., Stark, K. L., Maksymetz, J., Lin, H., Kaminker, J. S., Jung, M., Foreman, O., Tao, J., Ngu, H., Joseph, V., Roose-Girma, M., Tam, L., Lardell, S., Orrhult, L. S., Karila, P., … Hackos, D. H. (2023). Nav1.7 is essential for nociceptor action potentials in the mouse in a manner independent of endogenous opioids. Neuron, 111(17), 2642-2659.e13. https://doi.org/10.1016/j.neuron.2023.05.024
Sidders, B., Karlsson, A., Kitching, L., Torella, R., Karila, P., & Phelan, A. (2018). Network-Based Drug Discovery: Coupling Network Pharmacology with Phenotypic Screening for Neuronal Excitability. Journal of Molecular Biology, 430(18), 3005–3015. https://doi.org/10.1016/j.jmb.2018.07.016
Presenting Author
Lauri Louhivuori / Lydia Moll
Poster Authors
Lauri Louhivuori, Ph.D.
PhD
Orion Pharma
Lead Author
Lydia Moll
Cellectricon AB
Lead Author
William Hennah
PhD
Orion Corporation Orion Pharma
Lead Author
Anne Vuorenpää
Orion Pharma
Lead Author
Linnea Orrhult
PhD
Cellectricon AB
Lead Author
Johan Pihl
Cellectricon AB
Lead Author
Paul Karila
PhD
Cellectricon AB
Lead Author
Carina Stenfors
Orion Corporation
Lead Author
Topics
- Models: Chronic Pain - Neuropathic