Add a "safety lock" to gene and cell therapy

on August 8th, Ye Haifeng, a researcher at the School of Life Sciences and the Center for Medical Synthetic Biology research at East China Normal University, published their latest research results online in Science Advances, using the design ideas of synthetic biology to develop a safe and efficient gene control switch for the regulation of the natural green small molecule, clinically approved drug sodium aweiteate.
gene and cell therapy are at the forefront of innovative disease therapies and have become breakthroughs in the treatment of many diseases, which are expected to enhance clinical efficacy and improve the quality of life of patients. However, cell therapy is usually used only in severe or pre-treated patient groups, with very serious adverse events that limit the group of patients who can benefit. Therefore, how to achieve personalized, accurate treatment and the precise and controlled release of living cell therapy drugs is a difficult problem facing the medical, industrial and academic circles.
scientists have proposed a variety of safeguards for gene and cell therapy, most of the options currently available rely on small molecular compounds to regulate the output of therapeutic proteins. Ye Haifeng told China Science Daily that these small molecule compounds mainly include a variety of antibiotics, cosmetic ingredients, food additives, etc. , in the course of disease treatment side effects and long-term in patients, are not clinically certified. In this study, the researchers used the clinically approved drug sodium aweites, approved by the State Drug Administration of China and which can be used for cardiovascular disease treatment, to prevent thrombosis as a trigger switch for gene lines, to regulate the release of therapeutic drugs, and to have a superior clinical background, which can help accelerate the clinical transformation of gene and cell therapy.
's team successfully constructed a GENE switch (FAR switch) regulated by aweic acid/sodium aweites, including FAROFF switch (off) and FARON switch (on), using the transcription-regulating inhibitor PadR of the PadA gene in Bacillus spores 168, which was able to dissofidge this characteristic from its specific DNA binding sequence when acetic acid was present in the environment.
FAROFF switch (off) in this study consists of synthetic transcription-activated sub-aPadR (VP64-PadR) and induced starter PaPadR (OPadR) 6-PhCMVmin. When there is no sodium aweitate, the transcriptional activater aPadR specifically binds to the induced initiater PaPadR to initiate the downstream report gene expression, and when there is sodium aweitate, the transcription activater aPadR is immediately dissophed from the initiationer PadR, thereby turning off the downstream gene expression. The results showed that FAROFF switch showed good gene expression dynamics for sodium aweic acid/sodium aweic acid and sodium aweic acid tablets, including dependence on different drug concentrations, dependence on different drug incubation times, reversibleness, and adjustability in different mammalian cell lineages.addition, the researchers further designed and built the FARON switch, which consists of two components: the synthetic transcription suppressor iPadR (KRAB-PadR) and the induced promoter PiPadR (OPadR-PSV40-OPadR(RC). FARON switch works in contrast to FAROFF switch, where downstream reported gene expression is turned off when sodium aweicate is not present in the environment, and downstream reported gene expression is triggered when sodium aweicate is present. The results showed that FARON switch also showed good gene expression dynamics for sodium aweic acid/sodium aweites and sodium aweic acid tablets.the high degree of controllability shown by the FAR switch, the researchers applied the switch to three studies, further confirming the flexibility and application breadth of FAR switch.
(i) can be applied to CRISPR-Cas9/dCas9 systems regulated by sodium arweitic acid. CRISPR-Cas9/dCas9 systems have been widely used in surface genetic modification and fixed-point gene editing, but the continuous expression of cas9 proteins is at risk of off-targeting, with unpredictable side effects. Therefore, on the basis of FARON switch, the researchers designed and modified the oscic genetic modification system (PadRa for endogenetic gene activation device padRa and endogenetic gene suppressor PadRi) and gene editing system (PadRdel for gene deletion device) for sodium aveic acid regulation, and realized gene editing and oscic genetic exercise for sodium aveic acid regulation.
(ii) can be applied to high-order logic operations in single cells controlled by the joint control of sodium aweic acid and benzoic acid. Mammalian cells are loaded with programmable gene regulatory networks (FAR switch and Benzoic acid-inducible switch) that perform a variety of high-order logic operations, assemble biological computers, and enable complex human-computer interactions, with the promise of providing patients with tailored and accurate drug-giving measures for personalized and accurate medical treatment.
(iii) can be applied to long-term controlled genetically modified expression in mice. To achieve the application of FARON switch in future gene and cell therapies, the researchers will include HEKFAR-ON-SEAP, a stable cell strain of FARON switch, which is wrapped in microcapsules and transplanted into mice. The results showed that mice containing FARON switch, whether injected with sodium aweites in the abdominal cavity or oral sodium aweiteate tablets, were able to detect the expression of the reported gene alkaline phosphatase (SEAP) in the serum of mice, and the induced regulatory period of the drug was up to 15 days.
, a clinically approved FAR switch for the regulation of the drug sodium aweites, has further enriched the synthetic biology toolbox and provided a new device for manipulating genetic genes, said Ye Haifeng. This study demonstrates a safe and robust gene dynamic regulatory device that provides a new strategy for the dynamic intervention of precision medicine based on gene and cell therapy, and escorts the rapid development of precision medicine. This small molecule control switch for clinical drugs will greatly accelerate the pace of application of intelligent gene/cell drugs to clinical transformation. (Source: Qin Zhiwei, China Science Journal)
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