2020 Fiscal Year Research-status Report
Evaluation of an immunotherapy for chronic pain by expressing a single domain intrabody against the pain marker GCH1 in the rat dorsal root ganglion neurons
| Project/Area Number |
18K08853
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| Research Institution | Shiga University of Medical Science |
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Principal Investigator |
J・P Bellier 滋賀医科大学, 神経難病研究センター, 助教 (80346022)
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| Co-Investigator(Kenkyū-buntansha) |
守村 敏史 滋賀医科大学, 動物生命科学研究センター, 准教授 (20333338)
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| Project Period (FY) |
2018-04-01 – 2022-03-31
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| Keywords | GTP cyclohydrolase 1 / immunotherapy / BH4 / scFv / peripheral ChAT / acetylcholine / serotonin |
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| Outline of Annual Research Achievements |
Tetrahydrobiopterin (BH4) is required for the synthesis of the neurotransmitters serotonin and nitric oxide, which are involved in the modulation of the nociceptive neurotransmission. This project aims at developing immunotherapy against chronic neuropathic pain based on a recombinant single-chain antibody (scFv) to block GCH1 (GTP cyclohydrolase 1), the rate-limiting enzyme involved in BH4 synthesis.
Hybridomas synthesizing monoclonal antibodies able to block BH4 synthesis were obtained. cDNAs for light and heavy chains antibodies were cloned and sequenced. Plasmids encoding for recombinant scFv were constructed. The expression of recombinant scFv antibodies was evaluated in HEK-293 cell line and bacterial culture. Recombinant scFv antibodies were tested on tissues for specificity. Transfection of plasmid encoding scFv in primary Primary cell cultures of rat dorsal root ganglion (DRG) was not efficient for further analysis. An alternative cell culture model (THP-1 human monocytic leukemia cell line) was used to assess scFv expression and to test its effect on the metabolism of BH4, therefore the inhibitory activity of novel antibodies can be evaluated. scFv antibodies having tendencies to aggregates and novel construction were engineered to improve the solubility of the scFv. Bioinformatic analysis was performed to model interaction between scFv and its target, it brings evidence for specificity and mechanism of inhibition.
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| Current Status of Research Progress |
Current Status of Research Progress
3: Progress in research has been slightly delayed.
Reason
All planned experiments were implemented or are currently ongoing. Reaching the most challenging experimental part substantially slow down the progress of the work. Besides, the outbreak of coronavirus pandemic also affected the project progress. The production of new scFv constructions resolved concerns regarding antibody aggregation rns in the cytoplasm. Testing constructs using primary culture of DRG neurons was extremely challenging, for this reason an alternative cell culture model (cell line) was chosen, it provides more convenient and reliable model to optimize scFv cellular expression, and assess its inhibitory effect on the metabolism of BH4. Sensory intrinsic primary afferent neurons of the enteric nervous system are investigated as am alternative to DRG for in vivo targeting.
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| Strategy for Future Research Activity |
The project will continue to follow the general plan, which was to test the ability of the recombinant scFv to block BH4 in sensory neurons in vivo. While transfection in DRG appears challenging, transfection in the sensory IPAN cell of the enteric nervous system is planned. Expression of the recombinant scFv in the rat gut after in vivo transfection will be assessed using both immunofluorescence and western-blot analyses. Next, the ability of the scFv recombinant antibody to interfere with BH4 metabolism will be assessed by HPLC using electrochemical detection. Manuscripts describing the results obtained so far are in progress.
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| Causes of Carryover |
The reagent bought were all as planned in the initial experimental design. Whenever possible reagents were bought during commercial campaigns, and from companies selling at the best price, it resulted in a substantial saving. Challenging last steps of the project associated with the current pandemic have slowed down the work progress. Because of the outbreak of the coronavirus pandemic, presentations of the work at international meetings have been canceled. The remaining incurring amount of the previous fiscal year will be used this year according to the general plan of the initial proposal.
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