| 研究実績の概要 |
Enterohemorrhagic E. coli (EHEC) is one of the pathogenic E. coli capable of producing Shiga-like toxins (Stx). Antibiotics are not recommended as treatment for patients infected with EHEC strains because it will induce the release of Stx causing serious human gastrointestinal disease. Thus, there is no good treatment options as for now. This study aims to development a new therapeutic agent, termed Cas13a-stx, for treatment of EHEC infections by packaging CRISPR-Cas13a designed to target stx into E. coli phage capsid. Cas13a-stx can infect EHEC and sequence-specifically recognize stx gene, then promiscuously cleave surrounding ssRNA molecules, leading to decrement of toxin production and bacterial death.
To achieve the research aims, the following experiments are underway: 1) bacteriophages have been isolated from various sources and their lytic activities are checked against multiple species of E. coli including EHEC strains and clinical strains. 2) The Cas13a targeting stx is designed and 3) loaded into the capsid of the isolated phage candidate. 4) The effect of Cas13a-stx in protecting cell damage from Stx action is verified in vitro using Vero cells and 5) in vivo using mice model.
Currently, 5 EHEC strains have already been confirmed to carry stx gene and the candidate phage that can infect EHEC strains is successfully isolated from sewage water. Next, whole genome sequence in this phage will be determined before it was used to package Cas13a targeting stx.
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| 今後の研究の推進方策 |
The genome sequence of the candidate phage isolated from sewage water in Jichi Medical University will be determined and virulence-associated genes such as stx gene and endolysin will be deleted. Next, morphological characterization of the candidate phage will be performed using transmission electron microscopy (TEM) and the characterized phage will be loaded with the designed Cas13a targeting stx. Finally, cytotoxic effect and sequence-specific killing of Cas13a-stx will be evaluated using mammalian cell line (Vero cell) and mouse model, respectively.
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