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Awarded Amount$882,350DiseaseNTD (Chagas disease)InterventionDrugDevelopment StageTarget ValidationCollaboration PartnersNational Institute of Advanced Industrial Science and Technology , High Energy Accelerator Research Organization (KEK) , London School of Hygiene and Tropical Medicine(LSHTM) , Institute of Tropical Medicine (NEKKEN) Nagasaki University
Introduction and Background of the Project
Trypanosoma cruzi (Tc), the causative protozoan parasite of Chagas disease, does not contain the genetic machinery that facilitates an RNA interference response that has been widely used for gene specific inhibition of expression. This has been a limiting factor for studies on searching appropriate genes of Tc as targets for designing a new medicine. We have established a protocol for gene knockdown or knockout (KO) experiments aimed at candidate drug targets. Our system is an effective and high-throughput gene deletion system for Tc, which is achieved by combinational use of CRISPR/Cas9 genome editing and image-recognition technologies.
Identification of candidate target Tc proteins which are essential for amastigote stage parasites to proliferate or to survive.
We will undertake exhaustive KO campaign for about 1,000 Tc genes in the epimastigote stage to identify those essential for parasite survival. The essentiality of selected genes will further be evaluated by the same KO experiment in trypomastigote and amastigote stages by our established stage specific KO system in vitro and in vivo. After identification of the targets, we will examine drug-like active compounds from TCATc (GSK) for their effect on target gene overexpressed Tc to elucidate the pathogenicity requirement.
How can your partnership (project) address global health challenges?
More than 1 billion people are infected with one of the 20 NTDs listed by the World Health Organization (WHO). Despite the major achievement in medicine in the past 50 years, kinetoplastid diseases are still without adequate treatments. In order to achieve the ambitious goals of global NTD sustainable control and/or elimination new tools are still required.
Chagas Disease: About 6 million to 7 million people, mostly in Latin America, are estimated to be infected with Tc. More than 10,000 people die every year, and an estimated 100 million people are at risk of acquiring the disease. Only two drugs are currently available for Chagas disease, benznidazole and nifurtimox – both requiring a 60-90 day treatment period. The goal is to develop effective, well-tolerated, inexpensive treatments for indeterminate-chronic phase and acute stage infections that could be used in combination. Combination therapy can improve treatment efficacy, reduce dose levels and toxicity and prevent the potential development of resistance.
What sort of innovation are you bringing in your project?
We have developed an effective and high-throughput gene deletion system for Tc, which was achieved by combinational use of CRISPR/Cas9 genome editing and image-recognition technologies.
Role and Responsibility of Each Partner
NEKKEN will be responsibel to coordinate the study including communication within the partners and GHIT office. Regular and extra-regular reports will be made by the project coordinator at NEKKEN. Each partners will be responsible for the following items described under the original project plan.
1. Prioritization of candidate genes for KO.[NEKKEN]: Generate target list will be prepared using systematic review and a bioinformatics.
2. Gene KO and evaluation [AIST]: 1) First exhaustive gene KO in epimastigotes (EPIs) and 2) Evaluation of the first KOs in the TRYPO/AMA stage [AIST , NEKKEN and LSHTM].
3. Phenotypic screening of compounds using AMA-essential gene overexpression strains [LSHTM, NEKKEN]
4. Validation of AMA-essential genes in vivo [LSHTM]: Evaluation of, at least, 10 AMA-essential genes in an in vivo system.
5. Identification of genes associated with pathogenicity in vivo [NEKKEN, AIST]: Evaluation in vivo of, at least, 10 AMA-non-essential genes.
6. overall discussion on the progress. [NEKKEN, AIST, LSHTM, KEK]
1. Project objective
Trypanosoma cruzi (Tc), the causative protozoa of Chagas disease, does not contain the genetic machinery that facilitates an RNA interference response. This has been a limiting factor for studies on gene function and target validation because gene knockdown or knockout experiments aimed at candidate drug targets are deemed necessary for effective inhibitor development as a preliminary step in the drug development process. The mission of this project is overcoming this limiting factor by combining the technologies of the participating institutions. And the overall goal is to show candidates for the target molecules for further drug discovery steps.
2. Project design
This project was designed upon the following technical bases: CRISPR/Cas9-driven high-throughput gene knockout system in Tc, direct genetic manipulation of cultured extracellular amastigote (exAMA), inducive transgene expression system in Tc, and in vivo imaging of intra-mice Tcs. We planed the following two steps: undertaking an exhaustive knockout campaign for more than 500 Tc genes in epimastigote (EPI) stage to identify those essential for parasite survival, and evaluating the essentiality of selected genes in AMA/trypomastigote (TRYPO) stages. The suitability as a drug discovery target was also considered from the aspects of metabolic pathways, molecular characteristics, past drug discovery situations, etc.
3. Results, lessons learned
At first, we selected genes for knockout using the following criteria: belonging to central metabolism, expressed in AMA/TRYPO stages, not multiple copy genes, and not showing high homology to human orthologues. According to this list, we performed 552 gene knockouts on the EPI stage and found that the most knockouts affected the cell proliferation with abnormal cell morphologies. We then ranked these genes based on the rate of growth arrest, and 116 genes were selected for the further evaluation in AMA/TRYPO stage. From the gene knockout result in exAMA, we found that about one-third of the 116 genes were particularly important also in this life-cycle stage to be targeted for drug discovery. To consider effective progress of the target-directed drug discovery, it is very important for determining the target molecules that they are enzymes, they have high solubility, and their three-dimensional structures are already determined. Three enzymes in two metabolic pathways were thus determined as the candidates for the target molecules. For the best candidate among these, we have already constructed the overexpression system in bacterial hosts.
Besides this progress, this project generated the following outcomes that can serve as new tools in drug discovery for Chagas disease:
・Gene knockout database describing the knockout effect on proliferation activities and morphologies on 552 genes.
・New Tc-specific gene expression systems, one is for hyper-expression and another for stage and tissue specific expression.
・New Tc proliferation assay systems using innovative fluorometric enzymatic reaction.