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Identification of How Chemotherapy Drug Works Could Deliver Personalized Cancer Treatment
The chemotherapy drug decitabine is commonly used to treat patients with blood cancers, but its response rate is somewhat low. Researchers have now identified why this is the case, opening the door to more personalized cancer therapies for those with these types of cancers, and perhaps further afield. Researchers have identified the genetic and molecular mechanisms within cells that make the chemotherapy drug decitabine—used to treat patients with myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) —work for some patients but not others. The findings should assist clinicians in developing more patient-specific treatment strategies. The findings were published in the Proceedings of the National Academies of Science on March 30. The chemotherapy drug decitabine, also known by its brand name Dacogen, works by modifying our DNA that in turn switches on genes that stop the cancer cells from growing and replicating. However, decitabine’s response rate is somewhat low (showing improvement in just 30-35% of patients), which leaves something of a mystery as to why it works well for some patients but not for others. To find out why this happens, researchers from the KAIST investigated the molecular mediators that are involved with regulating the effects of the drug. Decitabine works to activate the production of endogenous retroviruses (ERVs), which in turn induces an immune response. ERVs are viruses that long ago inserted dormant copies of themselves into the human genome. Decitabine in essence, ‘reactivates’ these viral elements and produces double-stranded RNAs (dsRNAs) that the immune system views as a foreign body. “However, the mechanisms involved in this process, in particular how production and transport of these ERV dsRNAs were regulated within the cell were understudied,” said corresponding author Yoosik Kim, professor in the Department of Chemical and Biomolecular Engineering at KAIST. “So to explain why decitabine works in some patients but not others, we investigated what these molecular mechanisms were,” added Kim. To do so, the researchers used image-based RNA interference (RNAi) screening. This is a relatively new technique in which specific sequences within a genome are knocked out of action or “downregulated.” Large-scale screening, which can be performed in cultured cells or within live organisms, works to investigate the function of different genes. The KAIST researchers collaborated with the Institut Pasteur Korea to analyze the effect of downregulating genes that recognize ERV dsRNAs and could be involved in the cellular response to decitabine. From these initial screening results, they performed an even more detailed downregulation screening analysis. Through the screening, they were able to identify two particular gene sequences involved in the production of an RNA-binding protein called Staufen1 and the production of a strand of RNA that does not in turn produce any proteins called TINCR that play a key regulatory role in response to the drug. Staufen1 binds directly to dsRNAs and stabilizes them in concert with the TINCR. If a patient is not producing sufficient Staufen1 and TINCR, then the dsRNA viral mimics quickly degrade before the immune system can spot them. And, crucially for cancer therapy, this means that patients with lower expression (activation) of these sequences will show inferior response to decitabine. Indeed, the researchers confirmed that MDS/AML patients with low Staufen1 and TINCR expression did not benefit from decitabine therapy. “We can now isolate patients who will not benefit from the therapy and direct them to a different type of therapy,” said first author Yongsuk Ku. “This serves as an important step toward developing a patient-specific treatment cancer strategy.” As the researchers used patient samples taken from bone marrow, the next step will be to try to develop a testing method that can identify the problem from just blood samples, which are much easier to acquire from patients. The team plans to investigate if the analysis can be extended to patients with solid tumors in addition to those with blood cancers. -Profile Professor Yoosik Kim https://qcbio.kaist.ac.kr/ Department of Chemical and Biomolecular Engineering KAIST -Publication Noncanonical immune response to the inhibition of DNA methylation by Staufen1 via stabilization of endogenous retrovirus RNAs, PNAS
A powerful strategy for developing microbial cell factories by employing synthetic small RNAs
The current systems for the production of chemicals, fuels and materials heavily rely on the use of fossil resources. Due to the increasing concerns on climate change and other environmental problems, however, there has been much interest in developing biorefineries for the production of such chemicals, fuels and materials from renewable resources. For the biorefineries to be competitive with the traditional fossil resource-based refineries, development of high performance microorganisms is the most important as it will affect the overall economics of the process most significantly. Metabolic engineering, which can be defined as purposeful modification of cellular metabolic and regulatory networks with an aim to improve the production of a desired product, has been successfully employed to improve the performance of the cell. However, it is not trivial to engineer the cellular metabolism and regulatory circuits in the cell due to their high complexity. In metabolic engineering, it is important to find the genes that need to be amplified and attenuated in order to increase the product formation rate while minimizing the production of undesirable byproducts. Gene knock-out experiments are often performed to delete those metabolic fluxes that will consequently result in the increase of the desired product formation. However, gene knock-out experiments require much effort and time to perform, and are difficult to do for a large number of genes. Furthermore, the gene knock-out experiments performed in one strain cannot be transferred to another organism and thus the whole experimental process has to be repeated. This is a big problem in developing a high performance microbial cell factory because it is required to find the best platform strain among many different strains. Therefore, researchers have been eager to develop a strategy that allows rapid identification of multiple genes to be attenuated in multiple strains at the same time. A Korean research team led by Distinguished Professor Sang Yup Lee at the Department of Chemical and Biomolecular Engineering from the Korea Advanced Institute of Science and Technology (KAIST) reported the development of a strategy for efficiently developing microbial cell factories by employing synthetic small RNAs (sRNAs). They first reported the development of such system in Nature Biotechnology last February. This strategy of employing synthetic sRNAs in metabolic engineering has been receiving great interest worldwide as it allows easy, rapid, high-throughput, tunable, and un-doable knock-down of multiple genes in multiple strains at the same time. The research team published a paper online on August 8 as a cover page (September issue) in Nature Protocols, describing the detailed strategy and protocol to employ synthetic sRNAs for metabolic engineering. In this paper, researchers described the detailed step-by-step protocol for synthetic sRNA-based gene expression control, including the sRNA design principles. Tailor-made synthetic sRNAs can be easily manipulated by using conventional gene cloning method. The use of synthetic sRNAs for gene expression regulation provides several advantages such as portability, conditionality, and tunability in high-throughput experiments. Plasmid-based synthetic sRNA expression system does not leave any scar on the chromosome, and can be easily transferred to many other host strains to be examined. Thus, the construction of libraries and examination of different host strains are much easier than the conventional hard-coded gene manipulation systems. Also, the expression of genes can be conditionally repressed by controlling the production of synthetic sRNAs. Synthetic sRNAs possessing different repression efficiencies make it possible to finely tune the gene expression levels as well. Furthermore, synthetic sRNAs allow knock-down of the expression of essential genes, which was not possible by conventional gene knock-out experiments. Synthetic sRNAs can be utilized for diverse experiments where gene expression regulation is needed. One of promising applications is high-throughput screening of the target genes to be manipulated and multiple strains simultaneously to enhance the production of chemicals and materials of interest. Such simultaneous optimization of gene targets and strains has been one of the big challenges in metabolic engineering. Another application is to fine tune the expression of the screened genes for flux optimization, which would enhance chemical production further by balancing the flux between biomass formation and target chemical production. Synthetic sRNAs can also be applied to finely regulating genetic interactions in a circuit or network, which is essential in synthetic biology. Once a sRNA scaffold-harboring plasmid is constructed, tailor-made, synthetic sRNAs can be made within 3-4 days, followed by the desired application experiments. Dr. Eytan Zlotorynski, an editor at Nature Protocols, said "This paper describes the detailed protocol for the design and applications of synthetic sRNA. The method, which has many advantages, is likely to become common practice, and prove useful for metabolic engineering and synthetic biology studies." This paper published in Nature Protocols will be useful for all researchers in academia and industry who are interested in the use of synthetic sRNAs for fundamental and applied biological and biotechnological studies. This work was supported by the Technology Development Program to Solve Climate Changes on Systems Metabolic Engineering for Biorefineries (NRF-2012-C1AAA001-2012M1A2A2026556) and the Intelligent Synthetic Biology Center through the Global Frontier Project (2011-0031963) of the Ministry of Science, ICT and Future Planning through the National Research Foundation of Korea.
New BioFactory Technique Developed using sRNAs
Professor Sang Yup Lee - published on the online edition of Nature Biotechnology. “Expected as a new strategy for the bio industry that may replace the chemical industry.”- KAIST Chemical & Biomolecular engineering department’s Professor Sang Yup Lee and his team has developed a new technology that utilizes the synthetic small regulatory RNAs (sRNAs) to implement the BioFactory in a larger scale with more effectiveness. * BioFactory: Microbial-based production system which creates the desired compound in mass by manipulating the genes of the cell. In order to solve the problems of modern society, such as environmental pollution caused by the exhaustion of fossil fuels and usage of petrochemical products, an eco-friendly and sustainable bio industry is on the rise. BioFactory development technology has especially attracted the attention world-wide, with its ability to produce bio-energy, pharmaceuticals, eco-friendly materials and more. For the development of an excellent BioFactory, selection for the gene that produces the desired compounds must be accompanied by finding the microorganism with high production efficiency; however, the previous research method had a complicated and time-consuming problem of having to manipulate the genes of the microorganism one by one. Professor Sang Yup Lee’s research team, including Dr. Dokyun Na and Dr. Seung Min Yoo, has produced the synthetic sRNAs and utilized it to overcome the technical limitations mentioned above. In particular, unlike the existing method, this technology using synthetic sRNAs exhibits no strain specificity which can dramatically shorten the experiment that used to take months to just a few days. The research team applied the synthetic small regulatory RNA technology to the production of the tyrosine*, which is used as the precursor of the medicinal compound, and cadaverine**, widely utilized in a variety of petrochemical products, and has succeeded developing BioFactory with the world’s highest yield rate (21.9g /L, 12.6g / L each). *tyrosine: amino acid known to control stress and improve concentration **cadaverine: base material used in many petrochemical products, such as polyurethane Professor Sang Yup Lee highlighted the significance of this research: “it is expected the synthetic small regulatory RNA technology will stimulate the BioFactory development and also serve as a catalyst which can make the chemical industry, currently represented by its petroleum energy, transform into bio industry.” The study was carried out with the support of Global Frontier Project (Intelligent Bio-Systems Design and Synthesis Research Unit (Chief Seon Chang Kim)) and the findings have been published on January 20th in the online edition of the worldwide journal Nature Biotechnology.
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