2018 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 / acetylcholine / GCH1 / pChAT |
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| Outline of Annual Research Achievements |
BH4 is a cofactor required for the synthesis of serotonin and nitric oxide, two neurotransmitters that are involved in the modulation of the nociceptive neurotransmission. The biosynthetic pathway for BH4 involves GTP cyclohydrolase 1 (GCH1) which is the BH4 synthetic pathway rate-limiting enzyme and is also known as a marker for neuropathic pain. GCH1 activity is regulated through its regulatory protein GCH1 feedback regulatory protein (GFRP). The peripheral choline acetyltransferase, an isoform of the biosynthetic enzyme for the neurotransmitter acetylcholine (Ach) is expressed in the primary afferent neuron of the dorsal root ganglion. We have accumulated evidence that pChAT may modulate GCH1 activity through GFRP
In this context, blocking GCH1 activity in the sensory system using antibody, or levering the interaction between GCH1 and its regulatory protein GFRP might be a strategy to modulate nociceptive neurotransmission. This project aims at developing an immunotherapy against chronic neuropathic pain based on recombinant single chain antibody that may reduce tetrahydrobiopterin (BH4) synthesis (either by blocking GCH1 activity or its feedback regulation). We have produced a specific monoclonal antibody against that only recognizes the regulated form of GCH1 located in the sensory system. Our research objective is to clone the cDNA sequence of the monoclonal antibody and develop a single chain antibody, then assess its expression in the primary afferent neurons and its efficiency to block BH4 synthesis in the sensory system specifically.
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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 works are progressing rather smoothly, while others encountered inherent technical difficulties that slow down and delay the experimental research process.
Several clones of hybridoma producing antibodies against GCH1 were cultivated. The isotypes of the antibody produced by each clone were determined. 5’-RACE-PCR technology for cloning the unknown sequence of antibodies variable chain owas established and optimized to improve reliability during reverse-transcription and amplification steps, to minimize errors. The cDNA of light and heavy variable chains from a selected number of hybridoma were cloned using above 5’-RACE-PCR technology and sequenced. A plasmid construction where cDNA for the heavy and light chains are inserted in a plasmid together with a spacer to obtain a single chain of variable fragment is currently under assessment. In addition, we completed experiments for a manuscript describing a novel antiserum against human pChAT. When the antiserum was applied on sections of the human colon, it revealed the detailed organization of cholinergic system for the first time and demonstrated the existence of pChAT in human. This result indicates that pChAT might become another relevant target for in a strategy to develop immunotherapy against neuropathic pain.
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| Strategy for Future Research Activity |
The project will continue to follow the goal initially planned.
It includes the characterization of the single-chain antibody expressed in HEK293 cell line using both immunofluorescence and western- blot analyses, then testing the recombinant single chain antibody immunoreactivity against GCH1 on sections of rat DRG. Similar experiments will be done in HEK293 cell overexpressing GCH1, to assess whether the intrabody can block GCH1 activity. In the absence of inhibition, or the absence of immunoreactivity against GCH1, new recombinant antibodies will be cloned from others hybridoma, and an extensive characterization experiment will be performed to obtain the best inhibitory/immunoreactive recombinant antibody. Next, the recombinant antibody will be tested in primary DRG neurons after transfection. BH4 synthesis by DRG will be assessed by HPLC using electrochemical detection. In the same time, binding of the intrabody with GCH1 will be evaluated using co-immunoprecipitation and blue-native-PAGE. 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 intracellular BH4 level.
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| Causes of Carryover |
The reagent bought were all as planned in the initial experimental design. Whenever possible, we took advantage of commercial campaigns to purchased reagents with a significant discount.Publication of our work in an international journal did not require English correction, either publication fee; all resulting in a substantial saving. Planned travel at international conference was postponed, but should take place during the novel fiscal year.
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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