assafsfsaf
Salmonella is a facultative anaerobic gram-negative coryneform bacteria. Salmonella belongs to the Enterobacteriaceae family and is an important medical pathogen of humans and animals. In my country, food poisoning caused by salmonella accounts for 40% of bacterial food poisoning incidents. Salmonella forms a complex bacterial group consisting of two species and six subspecies, including more than 2,579 serotypes. Currently, two species of Salmonella have been identified, S. enterica and S. bongori. Salmonella's selective targeting of tumor tissues also makes it an ideal carrier for tumor targeted therapy. As an intracellular parasite, Salmonella has the characteristics of efficiently replicating in tumor tissues and effectively inhibiting tumor growth. After genetic engineering, it can be used as a carrier for tumor gene therapy in liver cancer, gastric cancer, colorectal cancer, etc. in vivo and in vitro.
Salmonella and Tumor Treatment
1. Attenuated Salmonella direct anti-tumor treatment:
Aiming at the characteristics of Salmonella that can effectively inhibit tumor growth, scientists have used a variety of genetic engineering techniques to modify the chromosomal genome of Salmonella, which can reduce the virulence of Salmonella, thereby obtaining attenuated strains. While reducing the pathogenicity to the host, Still let it retain a high degree of immunogenicity, thus ensuring the safety of clinical application.
Studies have shown that attenuated Salmonella has good targeted colonization and has a direct oncolytic effect on tumors. Using attenuated Salmonella to target and selectively replicate and proliferate in tumor tissues can effectively inhibit the growth of multiple tumor cells and prolong the survival time of animals.
2. As a tumor gene therapy carrier:
Attenuated Salmonella can carry foreign genes, cytokines, and foreign effector proteins to treat tumors. Cytokines can play an anti-tumor effect by directly killing tumor cells. The exogenous effector protein can be effectively delivered by attenuated Salmonella and express the therapeutic protein.
Salmonella knocks out the phoP gene to construct an attenuated Salmonella to improve the safety of Salmonella in treating tumors
Scientists used gene splicing PCR method combined with λ-Red system to delete the phoP gene of wild-type Salmonella typhimurium. This phoP gene is a transcriptional regulator, and it is also a component of a two-component regulatory system that plays a key role in biological adaptation to the internal environment and survival in macrophages. It also controls the expression of more than 40 genes required for the pathogenicity of Salmonella typhimurium, as well as resistance to the host’s adverse environment such as low pH in the stomach, bile salts, low oxygen in the small intestine, and cationic antimicrobial peptides on epithelial cells ability. The destruction of the phoP gene of Salmonella typhimurium prevents it from surviving in phagocytes and increases its sensitivity to host stress factors, thereby achieving attenuating effects in order to develop safer bacterial treatments.
The researchers used SOEing PCR and western blot methods to destroy the phoP gene of the wild-type and standard strains of Salmonella typhimurium. Three standard PCR and one fusion PCR reaction were used to construct linear DNA containing the upstream and downstream of the phoP gene and the kanamycin cassette. The pKD4 carrying the kanamycin gene was used as a template plasmid with FRT (FLP recognition target) sites on the side. The obtained structure was electroporated into competent cells of Typhimurium. After PCR verification, it was confirmed that the kanamycin gene had replaced the PhoP gene. Therefore, the attenuated Salmonella was successfully constructed by knocking out the Phop gene.
Gene editing bacteria brings unlimited possibilities to scientific research, clinical medicine, and agricultural industrial production!
Gene knockout has been used for many purposes, such as studying gene function, vaccine production, and improving the structure or expression of proteins. At present, the widely used traditional gene editing methods include the use of R6K suicide plasmid and the λ-Red system.
Although the λ-Red system seems simple and has been successfully applied to E. coli and other Gram-negative bacteria, due to the inherent differences of bacteria, the performance of this system in different bacteria is different. In addition, suicide plasmid vectors have disadvantages such as narrow host range and residual resistance. Traditional gene knock-out technology has disadvantages such as large post-screening workload (cycle), low recombination efficiency, and residual loxP or FRT sites.
CRISPR/Cas9 technology is the fastest-growing gene editing technology in recent years, but due to the lack of repair systems in bacteria, there are not many types of microorganisms that use this technology to edit genes. Yuanjing Biosciences has taken a different approach and created the original CRISPR-B™ technology. As an efficient genome editing technology, CRISPR-B™ technology has many advantages such as simple operation, strong targeting, low miss-target rate, and no trace. It can perform high-efficiency gene editing on various bacteria, and the efficiency is more than 20 times that of traditional methods. It can quickly achieve bacterial knockout, point mutation or knock-in.
Salmonella targets the shRNA expression of CTNNB1 gene to establish a new tool for tumor suppression
Bacterial therapy has good safety in the clinical treatment of gastrointestinal diseases such as diarrhea, irritable bowel syndrome, and inflammatory bowel disease. Various non-pathogenic anaerobes that can infiltrate and replicate in solid tumors can be used to develop therapies for human solid tumors. Ribonucleic acid interference (RNAi) has been established as an important research tool with great potential for gene therapy. Scientists have combined bacterial therapy and RNAi therapy to develop bacteria-mediated RNAi, which delivers shRNA-expressing vectors to target cells to silence disease-causing genes.
Compared with control cells, CTNNB1 gene silencing in SW480 cells can significantly reduce cell proliferation and death.
At the same time, the SW480 transplanted tumor model was constructed to verify the inhibition of tumor development by Salmonella-mediated CTNNB1 gene silencing in vivo. BALB/c female mice were randomly divided into three groups and received phosphate buffered saline (PBS), SL-Psls-TAT and SL-pSLS-huCAT respectively. Then record the growth of each group of tumors and the expression of CTNNB1 and its downstream target genes c-Myc and cyclin D1 in each tumor within two weeks. In the SW480 transplanted tumor model, compared with the PBS control group, the growth of transplanted tumors treated with SL-pSLS-huCAT was reduced by 65%, and the growth of transplanted tumors treated with SL-pSLS-TAT was reduced by 45%. At the same time, CTNNB1, c-Myc and cyclin D1 levels were reduced in these tumors. Therefore, SL-pSLS-CAT-mediated CTNNB1 knockdown significantly reduced tumor growth in SW480 transplanted mice. Subsequently, SL-pSLS-mCAT was orally administered to APCmin mice to test whether Salmonella expressing CTNNB1 shRNA can inhibit the growth of polyps. It was found that SL-pSLS-mCAT reduced the small intestine CTNNB1 mRNA level by 34%, polyps by 73% and mucosal tissue by 83%, compared with the SL-pLSLS-TAT injection group. SL-pSLS-CAT treatment also reduced the expression levels of these genes in mouse polyps, mucosal tissues and small intestine.
These data indicate that attenuated Salmonella expressing shRNA may be a powerful new tool for in vitro gene silencing, functional genomics and the development of RNAi-based anti-cancer or human immunodeficiency virus therapy.
Yuanjing Bio has rich experience in Salmonella gene editing and exogenous gene expression. The original CRISPR-B™ technology can perform efficient gene editing on various Salmonella bacteria. The efficiency is more than 20 times higher than that of traditional methods, and it can quickly achieve bacterial knocking. Except, point mutation or knock in! Contact us now to learn more about services related to your research!！
References:
1.A. Andino and I. Hanning.Review Article Salmonella enterica
: Survival, Colonization, and Virulence Differences among Serovars. 2015.e Scientifific World Journal.
3. Zhu Xiaozhou, Kong Guimei, Wan Dan, Sun Guozhuang, Jiao Hongmei, Yin Yinyan, Li Guocai. Research progress of attenuated salmonella in digestive system tumors. 2017. World Chinese Journal of Digestion. 1480-1485
4.H Guo, J Zhang and C Ina.Targeting tumor gene by shRNA-expressing Salmonella-mediated RNAi.Gene Therapy.2011.18:95-105
5.Ahani Azari, A. Zahraei Salehi, T. Nayeri Fasaei B. and Alebouyeh, M.. Gene disruption in Salmonella typhimurim by modified λ Red disruption system. Iranian Journal of Veterinary Research ,Shiraz University.2015.52:301-305
