[Introduction of "Access to Health" Initiatives for Neglected Tropical Diseases (NTDs)～R&D of NTD Medicines～].

Astellas regards the situation where there still remain barriers for many people who have difficulty accessing the healthcare they need due to the lack of available treatments, poverty, healthcare system challenges and insufficient healthcare information. Astellas recognizes this problem as the Access to Health issue. To improve Access to Health, Astellas has identified four areas where we can leverage our strengths and technologies, and is working to solve issues, making full use of external partnerships. These areas are "creating innovation", "enhancing availability", "strengthening healthcare system" and "improving health literacy". In March 2018, Astellas participated in the Neglected Tropical Diseases Drug Discovery Booster, a consortium whose purpose is to identify lead compounds for leishmaniasis and Chagas disease, both of which are neglected tropical diseases (NTDs). Moreover, within the pediatric praziquantel consortium, involving pharmaceutical companies, research institutions and international non-profit organizations, Astellas has developed a pediatric formulation of praziquantel for the treatment of schistosomiasis. In this report, we will introduce concretely how Astellas has promoted new drug development projects for NTDs in collaboration with various external partners, giving the projects as examples.

Development of a method to rapidly assess resistance/susceptibility of Micro-Tom tomatoes to Tomato yellow leaf curl virus via agroinoculation of cotyledons.

OBJECTIVE: Tomato yellow leaf curl virus (TYLCV) is one of the pathogens severely damaging tomato crops. Therefore, methods to treat or prevent TYLCV infection need to be developed. For this purpose, a method to conveniently and quickly assess infection of tomatoes by TYLCV is desired. In the present study, we established a quick method to evaluate TYLCV infection using cotyledons of Micro-Tom, a miniature tomato cultivar. RESULTS: First, we constructed a binary plasmid harboring 1.5 copies of the TYLCV genome and transformed Agrobacterium with the plasmid. By injecting agroinoculum from the resulting transformant into the branches of Micro-Tom, we confirmed the susceptibility of Micro-Tom to TYLCV. To shorten the evaluation process of TYLCV infection further, we agroinoculated cotyledons of Micro-Tom 10 days after sowing seeds. We consistently observed typical symptoms of TYLCV infection on true leaves 10 days after agroinoculation. Molecular analysis detected TYLCV progeny DNA in all leaves demonstrating symptoms 6 days after agroinoculation. Therefore, our new protocol enabled assessment of TYLCV infection within 20 days after sowing seeds. Thus, agroinoculation of Micro-Tom cotyledons will accelerate the process of screening TYLCV-resistant Micro-Toms and enable screening of larger numbers of plants more quickly, contributing to the development of TYLCV-resistant tomatoes.

Inhibition of binding of tomato yellow leaf curl virus rep to its replication origin by artificial zinc-finger protein.

Previously we demonstrated that inhibition of replication-associated protein (Rep) binding to its replication origin by artificial zinc-finger proteins (AZPs) is a powerful method to prevent plant virus infection in vivo. In the present study, we applied the AZP technology to Tomato yellow leaf curl virus (TYLCV), which is a limiting factor in tomato cultivation worldwide. First, we determined 5'-ATCGGTGT ATCGGTGT-3' in the 195-bp intergenic region of the TYLCV-Israel strain, a strain reported first among TYLCV strains, as the Rep-binding site by gel shift assays. We then constructed a 6-finger AZP that bound to a 19-bp DNA including the Rep-binding site. We demonstrated that the binding affinity of the AZP was >1,000-fold greater than that of Rep and that the AZP inhibited Rep binding completely in vitro. Because the binding capability of the AZP was same as that of the AZP previously designed for geminivirus-resistant Arabidopsis thaliana, we predict that the present AZP will prevent TYLCV infection in vivo.

Construction of plants resistant to TYLCV by using artificial zinc-finger proteins.

Previously, we have demonstrated that plant DNA virus replication could be inhibited in Arabidopsis thaliana by using an artificial zinc-finger protein (AZP) and created AZP-based transgenic A. thaliana resistant to DNA virus infection. Here we apply the AZP technology to tomato yellow leaf curl virus (TYLCV) causing serious damage to an important agricultural crop, tomato. An AZP was designed to block binding of the TYLCV replication protein (Rep) to the replication origin. The designed AZP had much higher affinities towards the replication origin than did the Rep, and efficiently blocked Rep binding in vitro. The AZP gene was then introduced into a plant genome with the help of Agrobacterium tumefaciens to generate the transgenic plants. The current status of the construction of the AZP-expressing transgenic plants will be reported.

Generation of plants resistant to tomato yellow leaf curl virus by using artificial zinc-finger proteins.

Previously, we designed an artificial zinc-finger protein (AZP) for blocking a replication protein (Rep) of beet severe curly top virus (BSCTV) from binding to its replication origin and demonstrated that transgenic Arabidopsis plants expressing the AZP are completely resistant to the virus infection. Here we applied the AZP technology to tomato yellow leaf curl virus (TYLCV) infective to an important agricultural crop, tomato. We designed and constructed an AZP binding to the direct repeat to block the TYLCV Rep binding. In gel shift assays, we confirmed that the designed AZP has a higher affinity to the replication origin than that of Rep and that the AZP effectively inhibited the Rep binding to its replication origin in vitro. The AZP gene was then introduced into a plant genome with the help of Agrobacterium tumefaciens to generate the transgenic plants. We will discuss properties of the AZP-transgenic plants against TYLCV infection.