WO2022094865A1

WO2022094865A1 - "autolytic" salmonella strain, preparation method therefor and application thereof in tumor treatment

Info

Publication number: WO2022094865A1
Application number: PCT/CN2020/126771
Authority: WO
Inventors: 刘陈立; 王作伟; 曾正阳; 盛方芊; 董宇轩; 卢伟琪; 郭旋
Original assignee: 中国科学院深圳先进技术研究院
Priority date: 2020-11-05
Filing date: 2020-11-05
Publication date: 2022-05-12

Abstract

Provided are a genetically engineered bacterium and a preparation method therefor. The genetically engineered bacterium is a salmonella strain that has knocked out dapA or dapE gene. Also provided is an application of the strain in tumor treatment.

Description

"Auto-lysing" Salmonella strain, its preparation method and its application in tumor therapy

technical field

The present invention relates to the fields of bioengineering and tumor treatment, in particular to a "self-lysing" Salmonella strain, its preparation method and its application in tumor treatment.

Background technique

Cancer is the leading cause of death worldwide. Compared with normal cells, cancer cells have the characteristics of infinite proliferation, transformation and easy metastasis. In addition to uncontrolled division (multipolar division), cancer cells also locally invade surrounding normal tissues and even metastasize to other organs via the circulatory system or lymphatic system in the body. The history of cancer treatment development shows that traditional cancer treatment methods, such as surgery, chemotherapy, radiation therapy, hormone therapy, bone marrow/stem cell transplantation, etc., all have certain defects, such as surgical treatment is prone to recurrence and some tumors are difficult to operate, etc. Problems; chemotherapy and radiotherapy can cause serious side effects to patients and make the treatment ineffective; hormone therapy tends to increase body fat mass and reduce muscle mass. This will affect blood sugar levels and increase the risk of diabetes; bone marrow/stem cell transplantation is difficult to match, and immune rejection is prone to occur. The difficulty of cancer treatment stems from its complex and changeable etiology. Not only changes at the gene level of the body, but also changes in the external environment are one of the important factors in the development of cancer. In the process of treating tumors with traditional methods, normal tissues and organs will be severely toxic, and cancer cells will develop multidrug resistance, which cannot completely eliminate cancer cells. In recent years, multiple studies have found that gene therapy, non-invasive radiofrequency treatment of cancer, insulin-boosting therapy, dietary therapy, and bacterial therapy can not only prevent cancer cells from developing multidrug resistance, but also enhance the efficacy of traditional therapies. Among them, bacterial therapy is a promising cancer treatment method that overcomes the shortcomings of traditional treatment methods.

The history of using live bacteria to treat cancer goes back more than 150 years. In 1868, the German physician W. Bush first applied bacteria to treat sarcoma that could not be treated by surgery. Within a week of receiving treatment, the tumor volume was reduced by half and the size of the cervical lymph nodes was reduced. Unfortunately, the patient died of sepsis due to bacterial infection 9 days later. In 1883, German surgeon Friedrich Fehleisen identified erysipelas as a cause of Streptococcus pyogenes infection. Subsequently, Friedrich Fehleisen and Willian B Coley, a surgeon from New York Hospital, independently conducted experiments to prove that Streptococcus pyogenes can make patients' tumors regress. However, the results were controversial because the experimental results were difficult to reproduce and did not meet the clinical standards of the time. In 1935 Connell observed that filtrates from the enzymes of Clostridium could cause metastases to regress. In 1947, scientists first injected spores of Clostridium histolytica into mice transplanted with sarcomas and observed lysis of cancer cells and regression of tumor tissue. However, the survival rate of mice was low due to the acute toxicity caused by the bacteria. In 1959, BCG (attenuated Mycobacterium bovine tuberculosis) was successfully used in cancer immunotherapy. In 2002, the attenuated Salmonella VNP20009 (msbB ^- , purI ^- ) underwent a phase I clinical trial, and the results showed that the strain could colonize tumor tissue, but the effect on tumor treatment was not obvious (Toso, JF et al. Phase I Study of the Intravenous Administration of Attenuated Salmonella typhimurium to Patients With Metastatic Melanoma. 20, 142-152, doi: 10.1200/jco.2002.20.1.142 (2002)).

Although VNP20009 did not achieve good clinical results, in view of the immunomodulatory function of Salmonella, researchers believe that various modifications may be used to make Salmonella suitable for tumor treatment. The reason why Salmonella needs to be modified is that wild-type Salmonella is virulent and can cause symptoms such as fever, vomiting, diarrhea, and abdominal cramps, and in severe cases, bacteremia can be life-threatening. With the rapid development of molecular biology technology, different strategies can be used to transform Salmonella to make it suitable for tumor therapy. It can knock out Salmonella and virulence-related genes, construct auxotrophic strains, regulate bacterial growth through gene circuits, etc., and promote the early use of attenuated strains for tumor treatment.

When Salmonella typhimurium was used in a mouse tumor model treatment test, it was found that the proliferation rate of wild-type Salmonella strains or auxotrophic Salmonella strains (eg: SL7207) in vivo was much greater than the clearance rate of the body, resulting in a large number of bacterial proliferation in mice. , causing severe bacteremia, resulting in lethal side effects (Yu, B. et al. Explicit hypoxia targeting with tumor suppression by creating an "obligate" anaerobic Salmonella Typhimurium strain. Sci Rep 2, 436, doi: 10.1038/srep00436 (2012). ).

Therefore, there is a need for Salmonella strains that have good tumor growth inhibition and are safer.

SUMMARY OF THE INVENTION

In order to solve the problems of the prior art, the purpose of the present invention is to provide an "autologous lysis" Salmonella strain, its preparation method and its application in tumor treatment.

In one aspect of the present invention, there is provided a method for obtaining a strain that cannot grow under both aerobic and anaerobic conditions by knocking out essential genes on the metabolic pathway of facultative anaerobic bacteria, and the strain is applied to in vivo tumors When treated, tumor growth can be inhibited and tumor volume reduced.

In one aspect of the invention, in the method, the facultative anaerobic bacterium is Salmonella spp.

In one aspect of the present invention, in the method, the strain needs to be additionally supplemented with 2,6-diaminopimelic acid (alias: 2,6-diaminoputaric acid; 2,6-diaminopimelic acid) when the strain is cultured in vitro. 6-Diaminopimelic acid) or its analogues.

In one aspect of the invention, in the method, the tumors and cancers include blood cancer (chronic leukemia, acute leukemia), bone cancer, lymphoma (non-Hodgkin's lymphoma, Hodgkin's lymphoma), bowel cancer (colon cancer, rectal cancer), liver cancer, stomach cancer, pelvic cancer (cervical cancer, ovarian cancer, endometrial cancer, ovarian cancer), lung cancer, breast cancer, pancreatic cancer, bladder cancer, prostate cancer, etc.

In one aspect of the invention, in the method, the bacterium is Salmonella typhi.

In one aspect of the invention, in the method, the facultative anaerobic bacterium is Salmonella typhimurium.

In one aspect of the present invention, in the method, the facultative anaerobic Salmonella strains include those derived from humans, chickens, dogs, cattle, and the like.

In one aspect of the present invention, in the method, the facultative anaerobic bacteria genus include: Enterobacteriaceae (Escherichia coli, Pneumococcus, Proteus, Enterobacter, Salmonella typhi, Salmonella, Shigella etc.), Staphylococcus, Streptococcus, Pneumococcus, Bacillus anthracis and Diphtheria, etc.

In one aspect of the present invention, there is provided a strain prepared according to the above method.

In one aspect of the present invention, there is provided a genetically engineered Salmonella strain in which the dapA or dapE gene is knocked out.

In one aspect of the present invention, there is provided a bacterial therapy of the above strain.

In one aspect of the present invention, the bacterial therapy is used in combination with other cancer treatment methods, including but not limited to surgery, radiotherapy, chemotherapy and the like.

In one aspect of the invention, the combination of other cancer treatment methods includes: (a) bacteriotherapy of the strain in combination with surgery; (b) bacteriotherapy of the strain in combination with radiation therapy; (c) bacteriotherapy of the strain in combination Chemical drugs: Chemotherapy drugs include alkylating agents (nimustine, carmustine, lomustine, cyclophosphamide, ifosfamide, mustard, etc.), antimetabolites (deoxyfluridine, etc.) , docefluridine, 6-mercaptopurine, cytarabine, fluoroguanosine, tegafur, gemcitabine, carmofur, hydroxyurea, methotrexate, eufodine, amcitabine, etc.), anti- Tumor antibiotics (actinomycin, arubicin, epirubicin, mitomycin, pelomycin, pingyangmycin, pirarubicin, etc.), plant anticancer drugs (irinotecan, three harringtonine, hydroxycamptothecin, vinorelbine, paclitaxel, taxotere, topotecan, vincristine, vindesine, vinblastine, etc.), hormones (atamestane, anastrozole, anlu Mitre, Letrozole, Formestane, Metgestrol, Tamoxifen, etc.) immunosuppressants and other anticancer drugs such as asparaginase, carboplatin, cisplatin, dacarbazine, oxaliplatin, Lexadine, Keplatin, Mitoxantrone, Procarbazine, etc.; (d) Bacteriotherapy combined with biological therapy of this strain; (e) Bacteriotherapy combined with traditional Chinese medicine treatment of this strain.

In one aspect of the present invention, there is provided the use of the above strain in in vitro inducing expression of a drug or as a carrier for carrying a drug, the drug being used for the treatment of cancer.

In one aspect of the present invention, in the application of the present invention, the medicament comprises: (a) expressing a protein substance or polypeptide substance with a cancer treatment effect; (b) expressing an RNA with a cancer treatment effect; (c) as a carrier Carrying modified RNA drugs.

Through the "autologous lysis" Salmonella strain of the present invention, its preparation method and its application in tumor treatment, a Salmonella strain with good tumor growth inhibiting ability and safer can be obtained.

Description of drawings

A to D in FIG. 1 are electropherograms of the construction of the SL7207 (ΔdapA) strain and the SL7207 (ΔdapE) strain according to the embodiment of the present invention.

Figures 2A to 2D: Figures 2A and 2B are the results of the in vitro experiments of the SL7207 (ΔdapA) strain according to an embodiment of the present invention, Figure 2A is a photo of the strain cultured under aerobic conditions for 1-10 days, and Figure 2B is a photo of the strain in anaerobic conditions Photos of culturing under aerobic conditions; Figure 2C and Figure 2D are the results of the in vitro experiments of the SL7207 (ΔdapE) strain according to an embodiment of the present invention, Figure 2C is a photo of the strain cultivated under aerobic conditions for 72 hours, and Figure 2D is a photo of the strain in anaerobic conditions Photos of 24h culture under conditions.

3A to 3C are the results of in vivo experiments of the SL7207 (ΔdapA) strain according to an embodiment of the present invention.

4A to 4C are the results of in vivo experiments of the SL7207 (ΔdapE) strain according to an embodiment of the present invention.

Detailed ways

While various modifications can be made to the invention and the invention can have various forms, specific examples will be described and explained in detail below. However, it should be understood that these are not intended to limit the present invention to the specific disclosure, and the present invention includes all modifications, equivalents or substitutes thereof without departing from the spirit and technical scope of the present invention.

In the following, "autolytic" Salmonella strains according to specific embodiments of the present invention, their preparation methods and their application in tumor therapy will be explained in more detail.

When Salmonella typhimurium was used in a mouse tumor model treatment test, it was found that the proliferation rate of wild-type Salmonella strains or auxotrophic Salmonella strains (eg: SL7207) in vivo was much greater than the clearance rate of the body, resulting in a large number of bacterial proliferation in mice. , causing severe bacteremia and lethal side effects. In this regard, the Salmonella typhimurium strain constructed by the present invention, which cannot survive under both aerobic and anaerobic conditions, is injected into tumor-bearing mice through tail vein, and 99% of the bacteria are eliminated by the body within one day. When the strain was applied to tumor therapy, it was found to have the ability to inhibit tumor growth for a long time, and the survival rate of mice was significantly increased. It shows that the present invention improves the safety on the basis of retaining the anti-tumor ability of bacteria.

The dapA or dapE gene is a key gene in the lysine metabolism pathway in bacteria and is involved in bacterial cell wall synthesis. When this gene is knocked out, the bacteria cannot synthesize diaminopimelic acid (DAP), the bacteria cannot form a complete cell wall, and the osmotic pressure inside and outside the bacteria is unbalanced, resulting in the bacteria being unable to survive. The invention utilizes the genetic engineering technology to knock out the essential gene dapA or dapE on the DAP (diaminopimelic acid) metabolic pathway to obtain the Salmonella typhimurium strain which cannot survive under both aerobic and anaerobic conditions.

By constructing a strain that can be easily eliminated by the body in a short time, the toxic and side effects to tumor-bearing mice due to the long-term persistence of bacteria in the body are reduced, and the modified strain is safer and more reliable, and does not affect the effect of bacteria on tumor treatment.

In one or more embodiments of the present invention, there is provided a method for obtaining a strain that cannot grow under both aerobic and anaerobic conditions by knocking out essential genes on metabolic pathways from facultative anaerobic bacteria, said When the strain is used for in vivo tumor treatment, it can inhibit tumor growth and reduce tumor volume.

The facultative anaerobic bacteria can be from Enterobacteriaceae bacteria (Escherichia coli, Pneumococcus, Proteus, Enterobacter, Typhoid Bacillus, Salmonella, Shigella, etc.), Staphylococcus, Streptococcus, Pneumococcus, Any species in any bacterial genus such as Bacillus anthracis and Bacillus diphtheriae.

The source of the facultative anaerobic Salmonella strain is not limited, as long as it is facultative anaerobic, for example, it includes facultative anaerobic Salmonella strains derived from humans, chickens, dogs, cattle and the like.

The facultative anaerobic bacterium is Salmonella typhimurium.

For example, the essential genes on the metabolic pathway are changed to dapA, dapB, dapD, argD, dapE, dapF, murE, murF, lysA, etc., preferably dapF, dapA, dapE genes. Especially the dapA or dapE genes.

When the essential gene dapA or dapE gene on the metabolic pathway is knocked out, the strain that cannot grow under both aerobic and anaerobic conditions needs to be additionally supplemented with 2,6-diaminopimelic acid (alias) when cultured in vitro. : 2,6-Diaminopimelic acid; 2,6-Diaminopimelic acid) or its analogs.

The strain of the present invention that cannot grow under both aerobic and anaerobic conditions can inhibit tumor growth and reduce tumor volume when applied to in vivo tumor treatment.

The tumor cancer includes blood cancer (chronic leukemia, acute leukemia), bone cancer, lymphoma (non-Hodgkin lymphoma, Hodgkin lymphoma), intestinal cancer (colon cancer, rectal cancer), liver cancer, stomach cancer, pelvic cancer (cervical cancer, ovarian cancer, endometrial cancer, ovarian cancer), lung cancer, breast cancer, pancreatic cancer, bladder cancer, prostate cancer, etc.

The present invention also provides bacterial therapy for the treatment of cancer using the strains of the present invention that cannot grow under both aerobic and anaerobic conditions.

The cancers include blood cancer (chronic leukemia, acute leukemia), bone cancer, lymphoma (non-Hodgkin lymphoma, Hodgkin lymphoma), bowel cancer (colon cancer, rectal cancer), liver cancer, stomach cancer, pelvic cancer ( Cervical cancer, ovarian malignant tumor, endometrial cancer, ovarian cancer), lung cancer, breast cancer, pancreatic cancer, bladder cancer, prostate cancer, etc.

The bacterial therapy of the present invention for the treatment of cancer can be used in combination with other cancer treatment methods.

The other cancer treatment methods used in combination include, but are not limited to, surgery, radiotherapy, chemotherapy, and the like.

The combined application of the bacterial therapy for treating cancer of the present invention and the other cancer therapeutic methods includes: (a) the bacterial therapy of the strain combined with surgical therapy; (b) the bacterial therapy of the bacterial strain combined with radiotherapy; (c) the bacterial therapy of the bacterial strain combined with radiotherapy; Bacterial therapy combined with chemical drugs: chemotherapy drugs include alkylating agents (nimustine, carmustine, lomustine, cyclophosphamide, ifosfamide, mustard, etc.), antimetabolites (deoxy Floxuridine, Docefluridine, 6-Mercaptopurine, Cytarabine, Flurguanosine, Tegafur, Gemcitabine, Carmofur, Hydroxyurea, Methotrexate, Eufodine, Amcitabine, etc. ), anti-tumor antibiotics (actinomycin, arubicin, epirubicin, mitomycin, pelomycin, pingyangmycin, pirarubicin, etc.), plant anticancer drugs (iritinib Kang, harringtonine, hydroxycamptothecin, vinorelbine, paclitaxel, taxotere, topotecan, vincristine, vindesine, vinblastine, etc.), hormones (atametan, anastrozole, etc.) , Anlutamide, Letrozole, Formestane, Metgestrol, Tamoxifen, etc.) Immunosuppressants and other anticancer drugs such as Asparaginase, Carboplatin, Cisplatin, Dacarbazine, Oxa Liplatin, Lexadine, Kebo Oxide, Mitoxantrone, Procarbazine, etc.; (d) Bacterial therapy of this strain combined with biological therapy; (e) Bacterial therapy of this strain combined with traditional Chinese medicine treatment.

The strains of the present invention that cannot grow under both aerobic and anaerobic conditions can also be used to induce expression of drugs in vitro or to carry drugs as carriers for cancer treatment.

In an embodiment of the present invention, the drugs that can be carried in the carrier include: (a) expressing a protein substance or polypeptide substance with a cancer treatment effect; (b) expressing an RNA with a cancer treatment effect; (c) as a carrier Carrying modified RNA drugs.

Example:

The following non-limiting examples are provided.

Example 1: Construction of SL7207 (ΔdapA) strain and SL7207 (ΔdapE) strain

The SL7207 (ΔdapA) strain and the SL7207 (ΔdapE) strain were constructed by λ-red homologous recombination technology. The specific steps are as follows:

(1) dapA-Up-HR-loxp-KnaR-loxp-dapA-Down-HR (HR: Homologous recombination arm) homologous recombination fragment, dapE-Up-HR- loxp-KnaR-loxp-dapE-Down-HR (HR:Homologous recombination arm) homologous recombination fragment 1;

(2) Prepare SL7207 electrotransformation competent cells, and transfer the pSim6 plasmid into the SL7207 strain by electrotransformation;

(3) Preparation of electrotransformed competent cells of SL7207 (pSim6) strain, and induced at 42°C for 15 min. The target fragment obtained in (1) was transferred into the SL7207 (pSim6) strain, and cultured overnight at 30°C on an LB solid plate (ampicillin + kanamycin + DAP);

(4) Colony PCR identifies positive clones that have undergone recombination;

(5) SL7207(ΔdapA)-loxp-KnaR-loxp-pSim6 strain and SL7207(ΔdapE)-loxp-KnaR-loxp-pSim6 strain were cultured overnight in LB (kanamycin+DAP) liquid medium at 37°C, The pSim6 plasmid was lost, and the SL7207(ΔdapA)-loxp-KnaR-loxp strain and the SL7207(ΔdapE)-loxp-KnaR-loxp strain were obtained;

(6) Preparation of SL7207(ΔdapA)-loxp-KnaR-loxp and SL7207(ΔdapE)-loxp-KnaR-loxp electrotransformation competent cells, and the Cre-705 plasmid was transferred into two target strains by electrotransformation. Homologous recombination with loxp removes the kanamycin resistance gene. Finally, the SL7207 (ΔdapA) strain and the SL7207 (ΔdapE) strain were obtained. The electropherograms of the construction of the SL7207 (ΔdapA) strain and the SL7207 (ΔdapE) strain according to the embodiment of the present invention are shown in FIG. 1 .

Table 1

Example 2: In vitro characterization of SL7207 (ΔdapA)

Characterization: The SL7207 (AdapA) strain containing kanamycin resistance was used for the test.

Characterization under aerobic conditions: Pick 3 single clones and resuspend in 10 μl LB medium respectively. 5 μl of bacterial resuspension was added to LB (DAP+) medium containing kanamycin and the remaining 5 μl of bacterial resuspension was added to LB (DAP-) medium containing kanamycin. The samples were taken out and photographed after 24 hours of incubation in an air shaker (37°C, 220 rpm). The bacteria grown in LB (DAP+) medium were re-inoculated into LB (DAP+) and LB (DAP-) medium containing kanamycin at a ratio of 1:100, cultured for 24 hours, and photographed for 10 consecutive days Uninterrupted transfer culture.

Characterization under anaerobic conditions: 3 single clones were picked and added to LB (DAP+) medium containing kanamycin. Incubate overnight in an air shaker (37°C, 220 rpm). The bacterial solution cultured overnight was put into an anaerobic incubator and transferred at a ratio of 1:100. Take 20 μl of the bacterial solution and add it to 2 ml of LB (DAP+) medium containing kanamycin; take 20 μl of the bacterial solution and add it to 2 ml of LB (DAP-) medium containing kanamycin, repeating 3 times. The initial OD600 value of the transferred samples was measured. In an anaerobic box, 37 ℃, static culture for 24h. Measure the OD600 value of the samples after 24h incubation.

Experimental results (shown in Figures 2A and 2B):

(1) Under aerobic conditions: the strain is cultured in LB(DAP+) and LB(DAP-) medium for 10 days without interruption, and can grow in LB(DAP+) medium; in LB(DAP-) medium unable to grow (Fig. 2A).

(2) Under anaerobic conditions: the strain was cultured in LB (DAP+) medium and LB (DAP-) medium for 24 hours. This strain could grow in LB(DAP+) medium; it could not grow in LB(DAP-) medium (Fig. 2B).

Experimental conclusion: The test of the strain under aerobic and anaerobic conditions showed that the facultative anaerobic strain SL7207 was successfully transformed into a strain that could not grow under both aerobic and anaerobic conditions.

Example 3: In vivo characterization of SL7207 (ΔdapA)

C57BL/6 mice were subcutaneously inoculated with 1×10 ⁶ mouse bladder cancer cells (MB49) to establish a mouse bladder cancer subcutaneous tumor model. The experiment was divided into three groups, PBS group, SL7207 strain group and SL7207(ΔdapA) group. The tail vein was inoculated with ¹ x 107 bacteria. The distribution of bacteria in normal tissues, organs and tumors of tumor-bearing mice, the changes in tumor volume, the changes in mouse body weight, and the survival rate of mice were detected. Experimental results (as shown in Figure 4A to Figure 4C ):

(1) Distribution of bacteria in tumor-bearing mice (Fig. 3A): The strains can be cleared from normal tissues and organs of mice within 7 days. In the SL7207 group, bacteria rapidly multiplied in normal tissues and tumors within 7 days, and finally all mice died within 7 days.

(2) Changes in tumor volume (Fig. 3B): Compared with the PBS group, this strain had an inhibitory effect on tumors during the experimental period.

(3) Changes in the body weight of mice ( FIG. 3B ): Compared with the PBS group, the mice in the SL7207 group lost more body weight; the mice in this strain group lost less body weight.

(4) Mice survival rate (Fig. 3C): During the experimental period, all mice in the SL7207 group died within 7 days; none of the mice in the PBS group and the strain group died.

Experimental conclusion: During the experimental period, (1) the tumor-bearing mice can clear the bacteria in the body within 7 days; (2) this strain has tumor-inhibiting effect; (3) the weight of mice injected with this strain is no higher than that of the PBS group. A very significant decrease, indicating that the strain of this strain is relatively less virulent, which improves the safety.

Example 4: In vitro characterization of SL7207 (ΔdapE)

Characterization: The SL7207 (AdapE) strain containing kanamycin resistance was used for the test.

Characterization under aerobic conditions: 3 clones were picked and added to 2 ml of LB (DAP+) medium containing kanamycin. Incubate overnight in an air shaker (37°C, 220 rpm). Transfer on the next day at a ratio of 1:100. 20 μl was added to 2 ml of kanamycin LB (DAP+) medium; 20 μl was added to 2 ml of kanamycin LB (DAP-) medium, 3 replicates. When cultured for 72h, the growth of the strain was observed and photographed.

Experimental results (as shown in Figures 2C and 2D):

(1) Under aerobic conditions: the strain was cultured in LB(DAP+) and LB(DAP-) medium for 72h, and it could grow in LB(DAP+) medium; it could not grow in LB(DAP-) medium ( Figure 2C).

(2) Under anaerobic conditions: the strain was cultured in LB (DAP+) medium and LB (DAP-) medium for 24 hours. This strain could grow in LB(DAP+) medium; it could not grow in LB(DAP-) medium (Fig. 2D).

Example 5: In vivo characterization of SL7207 (ΔdapE)

C57BL/6 mice were subcutaneously inoculated with 1×10 ⁶ mouse bladder cancer cells (MB49) to establish a mouse bladder cancer subcutaneous tumor model. The experiment was divided into two groups, PBS group and SL7207(ΔdapE) group. The tail vein was inoculated with ¹ x 107 bacteria. The distribution of bacteria in normal tissues, organs and tumors of tumor-bearing mice, the changes in tumor volume, the changes in mouse body weight, and the survival rate of mice were detected. Experimental results (as shown in Figure 4A to Figure 4C ):

(1) Changes in tumor volume (Fig. 4A): Compared with the PBS group, this strain had an inhibitory effect on tumors during the experimental period.

(2) Changes in the body weight of mice (Fig. 4B): Compared with the PBS group, there was no significant change in the body weight of mice injected with this strain.

(3) Mice survival rate (Fig. 4C): During the experimental period, neither the PBS group nor the strain group died.

Experimental conclusion: During the experimental period, (1) this strain has tumor-inhibiting effect; (2) the body weight of mice injected with this strain does not decrease significantly compared with the PBS group, indicating that this strain is relatively less toxic and improves safety.

Claims

A method for obtaining a strain that cannot grow under both aerobic and anaerobic conditions by knocking out essential genes in the metabolic pathway of facultative anaerobic bacteria, and the strain can inhibit tumor growth when applied to in vivo tumor and cancer treatment and reduce tumor volume.
The method according to claim 1, wherein the facultative anaerobic bacteria genus comprises: Enterobacteriaceae (Escherichia coli, Pneumococcus, Proteus, Enterobacter, Salmonella, Shigella, etc.) , Staphylococcus, Streptococcus, Pneumococcus, Bacillus anthracis and Diphtheria, etc.; and/or

Preferably, the facultative anaerobic bacteria are Salmonella; and/or

Preferably, the bacterium is Salmonella typhi; and/or

Preferably, the facultative anaerobic bacterium is Salmonella typhimurium; and/or

Preferably, the facultative anaerobic Salmonella strains are derived from humans, chickens, dogs, cattle and the like.
The method according to claim 1, wherein the strain needs to be additionally supplemented with 2,6-diaminopimelic acid (alias: 2,6-diaminopimelic acid; 2,6-Diaminopimelic acid) when the strain is cultured in vitro acid) or its analogs; and/or

The essential gene for the facultative anaerobe to be knocked out is the dapA or dapE gene.
The method of claim 1, wherein the tumors and cancers include blood cancer (chronic leukemia, acute leukemia), bone cancer, lymphoma (non-Hodgkin lymphoma, Hodgkin lymphoma), bowel cancer (colon cancer) , rectal cancer), liver cancer, stomach cancer, pelvic cancer (cervical cancer, ovarian cancer, endometrial cancer, ovarian cancer), lung cancer, breast cancer, pancreatic cancer, bladder cancer, prostate cancer, etc.
A strain prepared by the method according to any one of claims 1-4.
A genetically engineered Salmonella strain in which the dapA or dapE gene is knocked out.
A bacterial therapy of the bacterial strain prepared by the method of any one of claims 1-4 or the bacterial strain of claim 6,

Preferably, the bacterial therapy is used in combination with other cancer treatment methods, and the combined treatment methods include but are not limited to surgery, radiotherapy, chemotherapy and the like.
The bacterial therapy of claim 7, wherein the combination of other cancer treatment methods comprises: (a) bacterial therapy of the strain combined with surgery; (b) bacterial therapy of the bacterial strain combined with radiation therapy; (c) bacterial therapy of the bacterial strain Bacterial therapy combined with chemical drugs: chemotherapy drugs include alkylating agents (nimustine, carmustine, lomustine, cyclophosphamide, ifosfamide, mustard, etc.), antimetabolites (de Oxfluridine, Doxyfluridine, 6-Mercaptopurine, Cytarabine, Flurguanosine, Tegafur, Gemcitabine, Carmofur, Hydroxyurea, Methotrexate, Eufodine, Amcitabine etc.), antitumor antibiotics (actinomycin, arubicin, epirubicin, mitomycin, peclomycin, pingyangmycin, pirarubicin, etc.), plant anticancer drugs ( Rinotecan, harringtonine, hydroxycamptothecin, vinorelbine, paclitaxel, taxotere, topotecan, vincristine, vindesine, vinblastine, etc.), hormones (atametan, anastrox) azole, amlutamide, letrozole, formestane, methaprogesterone, tamoxifen, etc.) immunosuppressants and other anticancer drugs such as asparaginase, carboplatin, cisplatin, dacarbazine, Thaliplatin, Lexadine, Kebo Oxide, Mitoxantrone, Procarbazine, etc.; (d) Bacteriotherapy of this strain combined with biological therapy; (e) Bacteriotherapy of this strain combined with traditional Chinese medicine treatment.
Use of the strain prepared by the method of any one of claims 1 to 4 or the strain of claim 6 in in vitro inducing expression of a drug or as a carrier for carrying a drug, the drug being used for the treatment of cancer.
The application of the strain according to claim 9 in inducing the expression of a drug in vitro or as a carrier for carrying a drug, wherein the drug comprises: (a) expressing a protein substance or polypeptide substance with a cancer therapeutic effect; (b) expressing a cancer RNA for therapeutic effect; (c) as a carrier to carry the modified RNA drug.