Recombinant bacteria and uses thereof

A bacterial, cellular technology applied in the fields of microbiology and molecular biology to address issues such as easy bleeding or bruising, reduced resistance, and high initial response rates that do not translate into survival benefits

Pending Publication Date: 2021-11-16
NAT UNIV OF SINGAPORE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Unfortunately, the high initial response rate to first-line chemotherapy does not appear to translate into a survival benefit
In addition, there are many undesired side effects associated with chemotherapy, such as temporary hair loss, mouth sores, anemia (lower number of red blood cells, which may cause fatigue, dizziness, and shortness of breath), leukopenia (lower number of white blood cells, which may reduce the resistance), thrombocytopenia (decreased number of platelets, which may lead to easy bleeding or bruising), and gastrointestinal symptoms such as nausea, vomiting, and diarrhea

Method used

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  • Recombinant bacteria and uses thereof
  • Recombinant bacteria and uses thereof
  • Recombinant bacteria and uses thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0090] Example 1: High DUSP10 expression is associated with better overall survival in CRC patients

[0091] Recently, more and more studies have confirmed that the dysregulation of DUSP expression is related to various cancers, and it is often reported that the functional alteration of DUSP is related to the alteration of MAPK activity in tumors. Published CRC survival datasets from Marisa L et al. (PLoS Med.2013.10, e1001453) and The Cancer Genome Atlas (TCGA) (Nature 2012.487, 330-337) were analyzed in detail to investigate the relationship between DUSP10 levels and CRC patient survival. plausible relationship. Such as figure 2 As shown in the patient cohort from Marisa L et al., the recurrence-free survival (RFS) probability of patients with tumors with high DUSP10 expression tended to be slightly higher (P=0.1) ( figure 2 A, left panel). After stratifying samples for ERK expression, high DUSP10 in tumors was found to be associated with approximately 20% higher probab...

Embodiment 2

[0092] Example 2: Overexpression of DUSP10 in human CRC cells inhibits growth and metastasis of cancer cells

[0093] To confirm the tumor suppressor function of DUSP10, human CRC cell lines Caco2 and DLD1 stably overexpressing DUSP10 were generated for implantation as tumor xenografts in immunocompromised mice. The results showed that DUSP10 overexpression reduced tumor growth in vivo ( image 3 ). The key difference between these two cell lines is the presence of a KRAS mutation (G13D) in DLD1, whereas wild-type KRAS is found in Caco2. This suggests that overexpression of DUSP10 can effectively inhibit tumor cell growth regardless of KRAS status. Interestingly, overexpression of DUSP10 in HCT116 human CRC cells carrying a Kras mutation (G13D) caused a decrease in liver metastases ( Figure 4 ). In this experiment, HCT116 cells overexpressing DUSP10 or a control vector were injected into the spleen of immunocompromised NSGS mice for metastasis within 3 weeks. In the live...

Embodiment 3

[0094] Example 3: Development of Lactococcus lactis expressing fibronectin binding protein (Lactococcus lactis-Fnb-DUSP10) for delivery of DUSP10 expression plasmid

[0095] DNA plasmids carrying (1) fibronectin-binding protein (Fnb) from Staphylococcus aureus and (2) human DUSP10 gene insertion sequences were constructed by modifying the vector backbone pTRKH3-ermGFP (Addgene Company). Figure 5 ). The resulting DNA plasmid was transformed into Lactococcus lactis (ATCC) by electroporation and selected on the basis of erythromycin resistance. Single positive clones were subsequently isolated and stored in 20% sterile glycerol until required for use. For all subsequent infection experiments, three different controls were performed, including an untreated group, a group infected with Lactococcus lactis carrying the vector backbone ("Lactococcus lactis-vector"), and a group infected with Lactococcus lactis carrying only the Fnb gene insert. The vector backbone of the group of L...

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Abstract

The present disclosure provides a recombinant, probiotic lactic acid bacterium, wherein the bacterium comprises a non-replicating plasmid vector comprising (a) a tumor suppressor gene or anti-inflammatory gene operably linked to a first promoter that directs expression of the tumor suppressor gene or anti-inflammatory gene in a mammalian cell, and (b) an adhesin gene operably linked to a second promoter that directs expression of the adhesin gene in the bacterium. The present disclosure also provides uses of the recombinant bacterium, and methods of constructing the recombinant bacterium. The present disclosure also provides a method of treating cancer in a subject, wherein the method comprises administering a pharmaceutically effective amount of a recombinant, probiotic lactic acid bacterium, and the bacterium comprises a non-replicating plasmid vector comprising an adhesin gene operably linked to a promoter that directs expression of the adhesin gene in the bacterium.

Description

[0001] Cross References to Related Applications [0002] This application claims the benefit of priority to Singapore Patent Application No. 10201901828T filed on 28 February 2019, the contents of which are hereby incorporated by reference in their entirety for all purposes. technical field [0003] The present invention relates to microbiology and molecular biology, especially the development of recombinant bacteria and the use of recombinant bacteria for the treatment of cancer. Background technique [0004] Colorectal cancer, or cancer of the colon and rectum, is the third most common cancer worldwide and the most commonly diagnosed cancer in Singapore. [0005] Surgery is the main form of treatment for colorectal cancer. However, surgical resection of the bulky tumor left microscopic residual disease, which eventually led to recurrence. [0006] For stages II and III, radiation therapy may be added to help kill the cancer and shrink the tumor. Radiation may also be us...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K35/744A61P35/00A61P29/00
CPCA61K35/744A61P29/00A61P35/00C12N9/16C12Y301/03048C07K14/31C07K14/195C12N15/746C07K2319/00A61K38/164A61K39/3955C12Y301/03016
Inventor 章勇良方经文
Owner NAT UNIV OF SINGAPORE
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