2019 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, 神経難病研究センター, 助教 (20333338)
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| Project Period (FY) |
2018-04-01 – 2021-03-31
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| Keywords | Pain / GTP cyclohydrolase 1 / immunotherapy / BH4 / scFv / pChAT / 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 that are involved in the modulation of the nociceptive neurotransmission. This project aims at developing a immunotherapy against chronic neuropathic pain based on a recombinant single-chain antibody (scFv) to block the synthesis tetrahydrobiopterin (BH4).
Hybridomas synthesizing monoclonal antibody able to block BH4 synthesis were obtained. The cDNA of light and heavy chains of the monoclonal antibodies were cloned, sequenced and analyzed to engineer plasmids encoding for productive scFv. Recombinant scFv antibodies expression was evaluated in HEK-293 cell line. Recombinant scFv antibodies were tested on tissues for specificity. To assess the functional activity of the recombinant scfv antibodies, primary cell cultures of rat DRG were obtained for transfection with plasmid encoding scFv. Alternatively, a cell culture model was established to facilitate the assessment of the scFv expression and functional effect on BH4 metabolism. BH4 metabolism was investigated in this model, and it show that it can be a suitable alternative to test the blocking ability of novel antibodies directed against BH4 metabolism, which can be a novel target for immunotherapeutic strategy.
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| Current Status of Research Progress |
Current Status of Research Progress
2: Research has progressed on the whole more than it was originally planned.
Reason
All planned experiments were implemented or are currently ongoing. Some aspects of the experimental work are progressing rather smoothly. However, further technical optimizations are required on challenging aspect of the project, for instance to express a cytosolic antibody, or to develop suitable and high-throughput cell model system. New scFv constructions are under development to circumvent the trouble of antibody aggregation in the cytoplasm. Besides, the initial plan to assess svFc functionality on primary culture of DRG neurons turned to be quite challenging in term of obtaining sufficient amount, efficient transfection, and reproducible quality. Therefore we developed an alternative cell culture model using a commercial cell line. This approach is very suitable for the present project and has the advantage to provide suitable amount of cells, which are easier to transfect, and show a general better reliability than the primary cell culture of rat DRG.
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| Strategy for Future Research Activity |
The project will continue by following the goals initially planned. This includes the characterization of the novel optimized recombinant single-chain antibody expressed in HEK293 cell line using both immunofluorescence and western- blot analyses, and test its immunoreactivity on tissue sections of rat DRG. Next, the plasmid encoding for recombinant single-chain antibody will be transfected in the novel cell model that we have developed. The objectives of this experiment is to assess its ability to block BH4 synthesis. BH4 synthesis will be assessed by HPLC using electrochemical detection. Next, we are planning to express the single-chain antibody in vivo in the DRG of normal rat, where we will verify its level of expression and its effect on BH4 level, eventually its analgesic effect in neuropathic pain model.
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| Causes of Carryover |
All reagents were bought as planned in the initial experimental design. Whenever possible, we brought reagents from companies selling at the lowest price, and took advantage of commercial campaigns to purchased reagents with a significant discount; all resulting in a substantial saving. Developping an alternative in vitro model system also resulted in considerable saving.
Remaining incurring amount of the previous fiscal year will be used this year according to the plan of your initial proposal.
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Research Products
(6 results)
