# A Review of the Use of Native and Engineered Probiotics for Colorectal Cancer Therapy
## 3 Genetically Modified-Bacteria for Colorectal Cancer (CRC) Treatment
### Engineered Bacteria Treat CRC via Therapeutic Protein Delivery
#### 3.1.4 RNA Interference (RNAi) Molecules  
RNAi is an efficient gene-silencing mechanism, and the expression of various genes of interest can be effectively suppressed via RNAi. This makes it a potential tumor gene therapy tool via the silencing of particular genes associated with cancer induction. However, RNAi-mediated therapy in vivo faces the challenge of insufficient targeting [82; 83]. Based on this, bacteria-mediated RNAi therapy may provide a good alternative strategy due to the excellent tumor-targeting properties of bacteria. Bacteria-mediated RNAi therapy involves employing bacteria as a vector to transport therapeutic RNAi effectors, such as short hairpin RNA (shRNA), into target cells to silence specific mRNA [84]. An earlier study engineered _E. coli_ BL21 DE3 strains to produce a shRNA. This interfering shRNA expressed by engineered strains in vitro and in vivo significantly induced the silencing of the cancer gene CTNNB1 (catenin beta 1), whose overexpression activates the CTNNB1 pathway and induces colorectal carcinogenesis [85] (Figure 5). This is the first confirmation of the feasibility of targeting genes in mammalian cells with engineered bacteria-producing interfering RNAs. After that, [PERSON] et al. employed attenuated _S. typhimurium_ rather than _E. coli_ as a vector to deliver shRNAs. This strategy led to a significant reduction in CTNNB1 gene expression in vitro and in vivo as well. In addition, engineered-strain-mediated CTNNB1 knockdown remarkably inhibited tumor growth and reduced the number and size of polyps, respectively, in mice with SW480 xenograft tumors and APC\({}^{\text{Min}}\) mice [78]. Another example involves the delivery of shRNA by _S. typhimurium_ targeting the inhibin alpha subunit (sh-INHA). INHA protein is overexpressed in CRC cells and tissues compared with normal counterparts, which may contribute to the development of CRC. As a result, _S. typhimurium_/sh-INHA resulted in the downregulation of INHA  
Figure 4: Engineered bacteria harboring angiogenesis regulation proteins can inhibit the suppression of tumor angiogenesis via targeting and destroying blood vessels.  
expression and the activation of caspase, as well as the downregulation of the expression of B-cell lymphoma 2 (Bcl-2) and B-cell lymphoma-extra-large (Bcl-xL) in CRC cells [79]. The administration of _S. typhimurium_/sh-INHA suppressed tumor growth and extended the survival of mice bearing CT26 tumors [79]. Notably, one study focused on modulating immune responses for CRC treatment via RNAi. In this study, an engineered attenuated _S. typhimurium_ strain armed with an shRNA plasmid targeting indoleamine 2,3-dioxygenase (shIDO-ST) was developed. CRC cells were immunosuppressedby the overexpression of indoleamine 2,3-dioxygenase (IDO). The administration of shIDO-ST downregulated the expression of IDO in the TME and activated innate immunity, finally resulting in an attenuation of tumor growth in a murine CT26 and MC38 tumor model [86].