Genetically engineered bacteria: construction, performance evaluation, and applications in disease diagnosis and treatment

Abstract:

With the in-depth study of the relationship between intestinal microbiome and host and the rapid development of genetic engineering, the application of genetically engineered bacteria (GEB) in the medical field has become a research hotspot. GEB refer to the bacteria that have been genetically engineered to efficiently express exogenous proteins or compounds to achieve specific goals. Compared with traditional drugs, GEB have a variety of advantages. The construction process of GEB includes the selection of chassis, the acquisition of functional genes, and gene transfer and recombination. At present, the bacteria serving as GEB chassis can be classified into two categories:generally regarded as safe (GRAS) strains and commensal strains. The application of multi-omics facilitates the selection of chassis. Functional genes can be obtained by PCR, DNA synthesis, CRISPR-Cas9 or Red/ET recombination system according to their sizes. Heat shock and electroporation are widely used for plasmid transfer in bacteria. Homologous recombination can directly integrate the target gene into the host's chromosome. The encapsulation technology can improve GEB's survival rate and colonization ability, and synthetic gene circuits can make GEB intelligent. Functional stability, effectiveness, and safety are the general indicators for evaluating GEB. The instability of plasmid is an inherent defect of the GEB constructed by plasmid-mediated gene transfer, while the GEB constructed by gene integration have strong stability. In addition, to achieve long-term stable expression of functional genes, researchers need to evaluate and alleviate the impact of load. The efficacy and safety of GEB need to be evaluated in vitro, in animal models and clinical trials. There are some methods that have successfully achieved optimization of the above indicators. GEB have been widely used in the diagnosis and treatment of inflammatory diseases, tumors, metabolic diseases, infectious diseases, neurological diseases, and other diseases, playing a unique role. However, there are still problems regarding construction methods, performance evaluation and optimization, and large-scale production, which limit the clinical application. In this paper, we introduced the methods for construction and performance evaluation of GEB, summarized the application in disease diagnosis and treatment in recent years, pointed out the existing problems, and prospected the development of this field in the future.