CN109880849B - GHOST-shRNA expression vector compound targeting tumor-related macrophages and application thereof - Google Patents

Abstract

The invention relates to the field of tumor biotherapy, in particular to a GHOST-shRNA expression vector compound of targeted tumor-related macrophages and application thereof. The technical problem to be solved by the invention is that no effective treatment means for targeting tumor-related macrophages exists in the prior art. The technical scheme for solving the technical problems is to provide a GHOST-shRNA expression vector compound targeting tumor-related macrophages, namely bacterial GHOST loaded with shRNA eukaryotic expression vectors for inhibiting LAMP2a expression. The GHOST-shRNA expression vector compound can specifically, effectively and low-toxicity interfere the expression of LAMP2a in macrophages, can effectively treat macrophage high-infiltration tumors, and has good application prospect.

Description

GHOST-shRNA expression vector compound targeting tumor-associated macrophages and application thereof

Technical Field

The invention relates to the field of tumor biotherapy, in particular to a GHOST-shRNA expression vector compound of targeted tumor-related macrophages and application thereof.

Background

Macrophages that infiltrate in tumor tissue are called tumor-associated macrophages, and have a high degree of infiltration in a variety of tumors and are clinically directly related to the prognosis of a tumor. In thatThe degree of infiltration of tumor-related macrophages in breast cancer, prostate cancer, endometrial cancer, bladder cancer, kidney cancer, follicular lymphoma, malignant uveal melanoma, esophageal epithelial cancer or squamous cell carcinoma has an obvious positive correlation with poor prognosis. In the immunotherapy of tumor, tumor-associated macrophages are highly potential therapeutic targets, and researchers find that inhibitors of CSF-1/CSF-1R pathway can eliminate TAM and increase CD8 in mouse breast cancer and cervical cancer models ⁺ T cell activity, thereby inhibiting tumor growth; in contrast, inhibitors of the CSF-1/CSF-1R pathway can produce a significant reduction in tumor volume in the brain glioma model. In addition, there are also proposals for treating tumors by targeting tumor-associated macrophages in ovarian cancer, prostate cancer, breast cancer and melanoma. LAMP2a (lysosome-associated membrane protein type 2 a) is a target protein lysosome receptor for chaperone autophagy and is also a key effector protein for chaperone autophagy. Molecular chaperone autophagy is mainly characterized by selective degradation of target proteins compared with other autophagies, independent of vesicle transport of target proteins to the lysosome lumen, and thus specific alteration of cellular processes without organelle damage or cell death. Therefore, LAMP2a can play a role of a specific switch in the physiological behavior of cells, namely, the physiological behavior of cells is changed by specifically degrading certain kinases, nuclear transcription factors or protein inhibitors and the like; and can accurately identify some damaged proteins without influencing the functions of other normal proteins. Currently, the focus of research on LAMP2a and chaperone autophagy focuses on cell senescence, neurodegenerative diseases and their roles in tumor cells, and does not address this as a therapeutic target in tumor-associated macrophages.

Escherichia coli GHOST is artificially induced empty shell of Escherichia coli. The expression of the soluble protein E of the enterobacter bacteriophage phiX174 is induced in the escherichia coli, so that inner and outer membranes of the escherichia coli are mutually dissolved and transmembrane pores are formed, and due to the pressure of an inner cavity, a bacterial matrix can be sprayed to the outside of cells, and only a shell with a relatively complete structure is left, namely GHOST. Because the main component of the outer wall of the escherichia coli cell is lipopolysaccharide phosphate, phosphate groups in the lipopolysaccharide phosphate have negative charges; while the amine groups rich in the inner membrane have a positive charge. This difference in internal and external membrane potential ensures that the negatively charged DNA plasmid will only bind inside the bacterium, but not outside. When transfected, the bacterial GHOSTs are only taken up by cells with phagocytic function, while the phagocytic cells in the tumor microenvironment are mainly tumor-associated macrophages. After being phagocytized by cells, phosphatidylethanolamine in the escherichia coli cell membrane is mutually soluble with a vesicle membrane of a phagocytic vesicle so as to ensure that part of DNA plasmid carried by GHOST escapes to cytoplasm from the hydrolysis environment of the phagocytic vesicle, and the expression of exogenous genes is completed. The mutual solubility of the inner membrane and the outer membrane of the bacteria induced by the dissolved protein E can destroy lipoid A (lipid A) groups of Lipopolysaccharide (LPS) in the outer wall of the bacteria, influence the core structure of the LPS and reduce the immunogenicity of GHOST; meanwhile, the immunogenicity of GHOST can be greatly reduced by the dissipation of bacterial contents. This bacterial GHOST is much less toxic to macrophages than the commonly used cationic liposomes. Therefore, the Escherichia coli GHOST can specifically, efficiently and safely transfect macrophages.

Disclosure of Invention

The technical problem to be solved by the invention is that no effective treatment means for targeting tumor-related macrophages exists in the prior art. The technical scheme for solving the technical problems is to provide a GHOST-shRNA expression vector compound for targeting tumor-related macrophages. The GHOST-shRNA expression vector compound of the targeted tumor-related macrophage is bacterial GHOST loaded with shRNA eukaryotic expression vectors for inhibiting LAMP2a expression.

Wherein the shRNA eukaryotic expression vector for inhibiting LAMP2a expression in the GHOST-shRNA expression vector compound for targeting tumor-associated macrophages is a shRNA sequence capable of expressing a 9 th exon sequence aiming at Lamp2 a.

The shRNA template sequence in the shRNA eukaryotic expression vector in the GHOST-shRNA expression vector compound of the targeted tumor-associated macrophage is shown as at least one of SEQ No.1, SEQ No.2, SEQ No.3, SEQ No.4, SEQ No.5 or SEQ No. 6.

Wherein, the shRNA eukaryotic expression vector in the GHOST-shRNA expression vector compound of the targeted tumor-related macrophage is a plasmid vector.

Wherein the plasmid vector in the GHOST-shRNA expression vector compound targeting the tumor-related macrophages is at least one of pENTR/U6, pLKO.1, pGPU6, pGPH1 or pGenesil-2.

Wherein the bacterial GHOST in the GHOST-shRNA expression vector compound targeting the tumor-related macrophages is GHOST of at least one of escherichia coli, salmonella typhimurium, vibrio cholerae, bacillus pneumoniae, actinobacillus pleuritis or haemolyticus mannheimia.

Wherein the bacterial GHOST in the GHOST-shRNA expression vector compound targeting the tumor-related macrophage is the GHOST of Escherichia coli DH5 alpha.

Wherein, the proportion of the bacterial GHOST and shRNA eukaryotic expression vector in the GHOST-shRNA expression vector compound targeting the tumor-related macrophage is as follows: every 10 th ⁸ The escherichia coli GHOST is loaded with 0.3-10 mu g shRNA eukaryotic expression vector. Preferably, every 10 ⁸ The escherichia coli GHOST loads 1-5 mu g shRNA eukaryotic expression vector. More preferably, every 10 ⁸ Coli GHOST loaded 2 ug shRNA eukaryotic expression vector.

The shRNA eukaryotic expression vector for inhibiting LAMP2a expression in the GHOST-shRNA expression vector compound targeting the tumor-related macrophages is loaded in the bacterial GHOST through the potential difference between the inside and the outside of the bacterial GHOST.

The invention also provides application of the GHOST-shRNA expression vector compound targeting the tumor-related macrophages in preparing a medicament for treating macrophage high-infiltration type tumors.

Wherein the macrophage high-infiltration tumor in the application is at least one of breast cancer, prostatic cancer, endometrial cancer, bladder cancer, renal cancer, follicular lymphoma, malignant uveal melanoma, esophageal epithelial cancer or squamous cell carcinoma.

In addition, the invention further provides a medicine for treating macrophage high-infiltration type tumor. The medicine is a preparation prepared by adding pharmaceutically acceptable auxiliary materials or auxiliary components into the GHOST-shRNA expression vector compound of the targeted tumor-related macrophage. The macrophage high-infiltration tumor is at least one of breast cancer, prostate cancer, endometrial cancer, bladder cancer, kidney cancer, follicular lymphoma, malignant uveal melanoma, esophageal epithelial cancer or squamous cell carcinoma.

Furthermore, the preparation in the medicine for treating macrophage high infiltration type tumor is injection. The injection can be injection or freeze-dried injection.

The invention also provides a method for preparing the GHOST-shRNA expression vector compound targeting the tumor-related macrophage. The method comprises the following steps:

a. designing an shRNA sequence aiming at the Lamp2a, and constructing an expression vector capable of expressing the shRNA sequence;

b. and loading the constructed shRNA eukaryotic expression plasmid into bacterial GHOST to prepare the GHOST-shRNA expression vector compound.

The method has the beneficial effects that on the basis of finding that the high expression of LAMP2a in tumor-related macrophages indicates the poor prognosis of macrophage high-infiltration type tumors, the constructed shRNA eukaryotic expression plasmid aiming at LAMP2a is loaded into bacterial GHOST, and the GHOST-shRNA expression vector compound which aims at the tumor-related macrophages and aims at LAMP2a is prepared. Experiments prove that the GHOST-shRNA expression vector compound can specifically, effectively and low-toxicity interfere the expression of LAMP2a in macrophages, can effectively treat macrophage high-infiltration tumors, and has a good application prospect.

Drawings

FIG. 1: OD of GHOST of Escherichia coli ₆₀₀ Curve line. pBV220 E.coli is a control group without the protein E coding sequence. Data are mean ± standard deviation, representative experiment.

FIG. 2: concentration of unloaded DNA in the E.coli GHOST loading system. Data are mean ± standard deviation, representative experiment.

FIG. 3: different OD ₆₀₀ DNA load of E.coli GHOST. Data are mean ± standard deviation.

FIG. 4: the DNA-loaded E.coli GHOST was subjected to gel electrophoresis with an equal amount of plasmid DNA, lane 1 shows plasmid DNA, lane 2 shows GHOST loaded with DNA after 24 hours or 48 hours at 4 ℃, and lane 3 shows E.coli GHOST in lane 2 washed with HBSS-equilibrated solution.

FIG. 5: mouse macrophage cell line RAW264.7 and colon cancer cell line CT26 cells were transfected with empty escherichia coli GHOST without plasmid loading or escherichia coli GHOST with GFP eukaryotic expression plasmid loading, respectively, and the expression of cellular GFP was observed under a fluorescent microscope 72 hours after transfection. DAPI is nuclear staining; the scale bar is 100 μm; representative experiment.

FIG. 6: balb/c mice (1 OD per mouse) were transfected in vitro with CT26 cells, RAW264.7 cells, or intraperitoneally injected with saline, empty E.coli GHOST without plasmid, E.coli GHOST with GFP eukaryotic expression plasmid, respectively. After 72 hours, cells cultured in vitro or peritoneal lavage of mice were harvested and GFP expression was detected by flow cytometry. Representative experiment.

FIG. 7: mouse bone marrow-derived macrophages stimulated by tumor cell culture supernatants are transfected by escherichia coli GHOST loaded with three different shRNA eukaryotic expression plasmids targeting Lamp2 a. After 72 hours, the cells were harvested, whole proteins were extracted, and LAMP2a expression was detected by western blotting. Three shRNA clones are shown in lanes 2, 3 and 4, respectively.

FIG. 8: a: respectively transfecting the mouse bone marrow-derived macrophages stimulated by the tumor cell culture supernatant by using escherichia coli GHOST (sh-NC) loaded with the shRNA eukaryotic expression plasmid without specific targeting or escherichia coli GHOST (sh-L2 a) loaded with the shRNA eukaryotic expression plasmid with the target Lamp2 a. After 72 hours, cells are harvested, holoprotein is extracted, and the expression of LAMP2a is detected by western blotting. B: lamp2a mRNA expression in mouse bone marrow-derived macrophages as described in figure 8A was examined by qPCR. The internal reference is Actin; data are mean ± standard deviation; test with one-way ANOVA; * p <0.05, p <0.01, p <0.001, ns has no statistical significance.

FIG. 9: balb/c mice were injected with 1OD, 2OD, 5OD GHOST or Normal Saline (NS) in the tail vein, respectively, as controls. After 12 hours, 24 hours, and 48 hours, the mice were sacrificed, and the concentrations of IL-1. Beta. IL-6 and TNF-. Alpha. In the peripheral blood of the mice were measured by ELISA. Each group of mice was 3; data are mean ± standard deviation.

FIG. 10: the PyMT tumor-bearing mice are respectively injected with Normal Saline (NS), escherichia coli GHOST (sh-NC) loaded with shRNA eukaryotic expression plasmids without specific targeting or escherichia coli GHOST (sh-L2 a) loaded with shRNA eukaryotic expression plasmids with target Lamp2a by tail veins. 2OD GHOST per mouse was administered once every 3 days. Mice were sacrificed at successive time points and tumors were weighed. Each group of mice is 4-6, and the experiment is repeated for 2-4 times; data are mean ± standard deviation; test with one-way ANOVA; * p <0.05, p <0.01, p <0.001, ns has no statistical significance.

FIG. 11: after physiological saline (NS), escherichia coli GHOST (sh-NC) loaded with shRNA eukaryotic expression plasmid without specific targeting or escherichia coli GHOST (sh-L2 a) loaded with shRNA eukaryotic expression plasmid with target Lamp2a are respectively injected into the PyMT tumor-bearing mice through tail veins, the expression of LAMP2a in tumor-related macrophages is detected by flow cytometry. Data are mean ± standard deviation; test with one-way ANOVA; * p <0.05, p <0.01, p <0.001, ns has no statistical significance.

FIG. 12: LAMP2a expression was detected by flow cytometry in PyMT mouse tumor infiltrating cells as described in figure 10. Data are mean ± standard deviation; test by one-way ANOVA; * p <0.05, p <0.01, p <0.001, ns are not statistically significant.

FIG. 13: the tumor volume of a Balb/c mouse inoculated subcutaneously with a mouse colon cancer cell line CT26 or a breast cancer cell line 4T1 is measured after intratumoral injection (i.t.) or tail vein injection (i.v.) of physiological saline (NS), escherichia coli GHOST (sh-NC) loaded with shRNA eukaryotic expression plasmid without specific targeting or escherichia coli GHOST (sh-L2 a) loaded with shRNA eukaryotic expression plasmid targeting Lamp2 a. 2OD GHOST per mouse were administered every 3 days. Tumor volume was calculated as (long diameter x short diameter) ² ) 2; each group of mice contains 5-8 mice, and the number of mice is 2Testing; data are mean ± standard deviation; test with one-way ANOVA; * p is a radical of<0.05,**p<0.01,***p<0.001,ns is not statistically significant.

FIG. 14: LAMP2a expression was detected by flow cytometry in PyMT mouse tumor infiltrating cells as described in figure 12. Data are mean ± standard deviation; test by one-way ANOVA; * p <0.05, p <0.01, p <0.001, ns has no statistical significance.

FIG. 15 tumor samples of 145 breast cancer patients were grouped and ranked for the amount of LAMP2a expression in tumor cells and tumor-associated macrophages, respectively, and their respective survival curves were counted and examined by Log-rank (Mantel-Cox).

Detailed Description

The present invention will be described in more detail with reference to the following embodiments.

According to the invention, clinical samples of 145 breast cancer patients are researched in the early stage, and the finding that the high expression of LAMP2a in tumor-associated macrophages indicates poor prognosis, while the expression of LAMP2a in tumor cells has no obvious correlation with the survival period of the patients. Since breast cancer is a typical macrophage highly-invasive tumor, the degree of macrophage infiltration in various solid tumors is obviously related to poor prognosis, such as breast cancer, prostate cancer, endometrial cancer, bladder cancer, kidney cancer, follicular lymphoma, malignant uveal melanoma, esophageal epithelial cancer or squamous cell carcinoma. Considering that the number of tumor cells is absolutely dominant in tumor tissues, the specific inhibition of the expression of LAMP2a in tumor-associated macrophages is probably a key problem in the targeted therapy related to macrophage high-infiltration type tumors.

Although, the inventors have not found a technique for interfering gene expression by delivering an shRNA vector in vitro and in vivo using E.coli GHOST as a vector. However, the inventor considers whether it is possible to use bacterial GHOST to load an shRNA expression vector for inhibiting LAMP2a expression and target tumor-related macrophages, so that the LAMP2a expression in the tumor-related macrophages is reduced, and the purpose of treating macrophage high-infiltration type tumors is achieved.

On the basis, the GHOST-shRNA expression vector compound targeting tumor-associated macrophages is developed. Namely bacterial GHOST loaded with shRNA eukaryotic expression vector for inhibiting LAMP2a expression.

In one embodiment of the invention, the shRNA eukaryotic expression vector for inhibiting LAMP2a expression in the GHOST-shRNA expression vector complex targeting tumor-associated macrophages is a shRNA sequence capable of expressing a 9 th exon sequence for LAMP2 a.

And the 9 th exon sequence of mouse Lamp2a (mouse NM _ 001017959.2) is an subtype a specific sequence (encoding a target protein recognition binding peptide segment required by the function of LAMP2 a) and is also required by the function of LAMP2a protein, so that shRNA designed according to the 9 th exon sequence of mouse Lamp2a can effectively inhibit the expression of LAMP2a and realize the corresponding function. Furthermore, the exon 9 sequence of Lamp2a is a highly conserved sequence, for example, the sequence similarity between mouse and human (NM-002294.2) reaches 91%. Therefore, aiming at the segment of the sequence, the GHOST-shRNA expression vector compound targeting the tumor-associated macrophages can achieve good curative effect on mice, and the technical scheme of the invention can also achieve good effect in treatment of human-associated tumors.

The template sequence of shRNA designed by the invention is shown as SEQ ID No.1, SEQ ID No.2 or SEQ ID No. 3. In the experiment, the effect is shown, wherein the effect of SEQ ID No.2 is better. Obviously, other different shRNA interfering molecules against exon 9 sequences of Lamp2a can also be designed. The invention also provides shRNA template sequences corresponding to the three mouse shRNA template sequences in the table 1 and aiming at corresponding sites of human Lamp2a, and the shRNA template sequences are shown as SEQ ID No.4, SEQ ID No.5 or SEQ ID No. 6.

In general, the shRNA eukaryotic expression vector used in the GHOST-shRNA expression vector complex targeting tumor-associated macrophages of the present invention may use a vector that can be loaded into bacterial GHOST, and a plasmid vector is generally recommended. In one embodiment of the invention, a commercial plasmid vector pENTR/U6 is used. Other shRNA eukaryotic expression plasmids commonly used in the field can also construct shRNA plasmids targeting Lamp2a and load the shRNA plasmids into escherichia coli GHOST. For example, plasmid vectors such as pLKO.1, pGPU6, pGPH1, pGenesil-2 and the like can be used.

And the bacterial GHOST part in the GHOST-shRNA expression vector compound for targeting the tumor-related macrophage. Since the preparation of bacterial GHOST has been reported, the skilled person can easily obtain the bacterial GHOST available. For purposes of the present invention, GHOST from bacteria such as Escherichia coli, salmonella typhimurium, vibrio cholerae, klebsiella pneumoniae, actinobacillus pleuriseus, or Mannheim haemolyticum are all contemplated means for loading and delivering shRNA expression vectors. In the examples of the present invention, GHOST of Escherichia coli DH 5. Alpha. Was used.

The shRNA eukaryotic expression vector for inhibiting LAMP2a expression in the GHOST-shRNA expression vector compound targeting the tumor-related macrophages is loaded in the bacterial GHOST through the potential difference between the inside and the outside of the bacterial GHOST.

Generally, in order to achieve a good target transfection effect, the bacterial GHOST is loaded with some shRNA expression vectors as much as possible.

A specific loading method is also presented in one embodiment of the invention to facilitate the practice of the invention. It is apparent that other means of loading shRNA expression vectors into bacterial GHOST may be used in the art to practice the present invention.

Currently, the ratio between shRNA expression vectors loaded by bacterial GHOST is: every 10 th ⁸ The escherichia coli GHOST is loaded with 0.3-10 mu g shRNA eukaryotic expression vector. Preferably, every 10 ⁸ Each escherichia coli GHOST loads 1-5 mu g shRNA eukaryotic expression vector. More preferably, every 10 ⁸ Coli GHOST loaded 2 ug shRNA eukaryotic expression vector.

Based on the scheme, the invention also provides application of the GHOST-shRNA expression vector compound of the targeted tumor-related macrophages in preparation of a medicine for treating macrophage high-infiltration type tumors.

Wherein the macrophage high-infiltration tumor in the application is at least one of breast cancer, prostatic cancer, endometrial cancer, bladder cancer, renal cancer, follicular lymphoma, malignant uveal melanoma, esophageal epithelial cancer or squamous cell carcinoma.

In addition, the invention further provides a medicine for treating macrophage high-infiltration type tumor. The medicine is a preparation prepared by adding pharmaceutically acceptable auxiliary materials or auxiliary components into the GHOST-shRNA expression vector compound of the targeted tumor-related macrophage. The macrophage high-infiltration type tumor is at least one of breast cancer, prostate cancer, endometrial cancer, bladder cancer, kidney cancer, follicular lymphoma, malignant uveal melanoma, esophageal epithelial cancer or squamous cell carcinoma.

Furthermore, the preparation in the medicine for treating macrophage high infiltration type tumor is an injection.

The term "pharmaceutically acceptable" means that the carrier, cargo, diluent, adjuvant, and/or salt formed is generally chemically or physically compatible with the other ingredients comprising a pharmaceutical dosage form and physiologically compatible with the recipient.

The mode of administration of the drug of the present invention is not particularly limited, and representative modes of administration include, but are not limited to: injection (intravenous, intramuscular, subcutaneous or intratumoral) and topical administration.

Compositions for injection may comprise physiologically acceptable sterile aqueous or anhydrous solutions, dispersions, suspensions or emulsions, and powders for reconstitution into sterile injectable solutions or dispersions. Suitable aqueous and nonaqueous carriers, diluents, solvents or vehicles include water, ethanol, polyols and suitable mixtures thereof.

Dosage forms for topical administration of the compounds of the present invention include ointments, powders, patches, sprays, and inhalants. The active ingredient is mixed under sterile conditions with a physiologically acceptable carrier and any preservatives, buffers, or propellants which may be required if necessary.

The pharmaceutically acceptable auxiliary material of the invention refers to a substance contained in a dosage form except for an active ingredient.

The pharmaceutically acceptable auxiliary components have certain physiological activity, but the addition of the components does not change the dominant position of the pharmaceutical composition in the disease treatment process, but only plays auxiliary effects, and the auxiliary effects are only the utilization of the known activity of the components and are auxiliary treatment modes which are commonly used in the field of medicine. If the auxiliary components are used in combination with the pharmaceutical composition of the present invention, the protection scope of the present invention should still be included.

The invention also provides a method for preparing the GHOST-shRNA expression vector compound targeting the tumor-related macrophage. The method comprises the following steps:

a. designing an shRNA sequence aiming at Lamp2a, and constructing an expression vector capable of expressing the shRNA sequence;

b. and loading the constructed shRNA expression vector into bacterial GHOST to prepare a GHOST-shRNA expression vector compound.

In one embodiment of the present invention, a plasmid is used as an expression vector. Dissolving the expression vector plasmid into sterile HBSS equilibrium liquid, then suspending lyophilized Escherichia coli GHOST by the HBSS equilibrium liquid, and shaking at 24 deg.C for more than 30 min to complete loading. The loading of the plasmid depends on the DNA concentration in the system and the number of GHOSTs. In this example, about 1 E.coli can carry about 6000 plasmids. After loading is complete, the unloaded expression vector plasmid is removed and the GHOST-shRNA expression vector complex is stored in sterile buffer or lyophilized for use. The loaded expression vector plasmid can be stably combined in GHOST, and the obvious loss can not be caused by multiple centrifugal washing or long-time standing.

Obviously, in addition to the HBSS equilibrium solution, other equilibrium solutions with ionic buffering effect can be used to complete the loading of the expression vector, such as PBS buffer solution.

The following examples are provided only for illustrating and confirming the present invention, and not for limiting the scope of application of the present invention. The protocols not specifically described in the examples below were performed according to conventional methods or the corresponding instructions provided by the reagent manufacturer.

Example 1 preparation of E.coli GHOST and Loading of DNA of interest

In order to facilitate the practice of the present invention, this example provides a specific method for preparing E.coli GHOST and loading the DNA of interest. Obviously, the preparation of E.coli GHOST and the loading of the target DNA can be realized by referring to other techniques reported in the prior art.

1) Constructing a GHOST induction plasmid: the coding sequence (GQ 153915.1) of phage phiX174 dissolving protein E is searched in NCBI database, the sequence is chemically synthesized, enzyme cutting sites EcoR I and Pst I are constructed at both ends of the sequence, the synthesized sequence and pBV220 plasmid are recombined into pBV220-Lysis E plasmid, and the recombined plasmid is transferred into DH5 alpha colibacillus. Besides the pBV220 plasmid used in the experiment, other prokaryotic expression plasmids with conditional promoter elements can theoretically realize the induction preparation of the GHOST of the escherichia coli.

2) Preparation of E.coli GHOST: culturing the recombinant engineering Escherichia coli in LB culture medium containing 100. Mu.g/mL ampicillin, and performing amplification culture at 28 deg.C and 220 rpm; when the bacterial concentration reaches about OD ₆₀₀ Approximately 0.6, the culture temperature is rapidly raised to 42 ℃ at which time the E.coli content begins to escape into the culture medium with activation of the dissolved protein E, the bacterial OD ₆₀₀ It continues to drop to about 0.1 to 0.2 as shown in fig. 1. After the induction was completed, escherichia coli GHOST was harvested by centrifugation at 8000g at 4 ℃. Then GHOST was resuspended in HBSS equilibrium containing 30mg/mL streptomycin, 34mg/mL chloramphenicol, and 50mg/mL kanamycin and left to stand at 4 ℃ overnight to kill unlysed E.coli. Afterwards, the GHOST was washed with sterile HBSS equilibration solution and stored by lyophilization. For convenience of describing the amount of GHOST, we will refer to 1OD as about 5X 10 hereinafter ⁸ Coli GHOST.

3) Loading of the DNA plasmid of interest: to facilitate the practice of the present invention, the present invention provides a specific method for loading a DNA plasmid into bacterial GHOST. First, the DNA plasmid to be loaded is dissolved in a solution containing 10mM sodium acetate, 25mM CaCl ₂ And 10mM HEPES, as much as possible to make the DNA concentration greater than 5mg/mL to ensure the loading efficiency. The lyophilized E.coli GHOST (recommended 100. Mu.L H) was then resuspended in this HBSS equilibriumBSS balance solution is re-suspended at about 30 to 60OD GHOST), and is shaken for more than 30 minutes at a constant temperature of 24 ℃. To examine the progress of plasmid loading, the loading system was centrifuged at 16,000g and the DNA content of the supernatant was measured, as shown in FIG. 2. The plasmid loading depends on the concentration of DNA in the system and the number of GHOSTs, and in our experiments the average loading of 1OD GHOST was about 10. Mu.g DNA (2000 to 3000bp plasmid) as shown in FIG. 3. About 6000 plasmids can be carried by 1 E.coli GHOST. After the loading was completed, GHOST was washed with sterile HBSS to remove unloaded DNA plasmid, and was stored in sterile HBSS at a concentration of 0.1 OD/. Mu.L at 4 ℃. The loaded DNA plasmid can be stably combined in GHOST, and the loss of the loaded DNA can not be caused by multiple centrifugal washing or long-time standing. If it is desired to determine the amount of DNA loaded by GHOST, the plasmid can be separated from GHOST by gel electrophoresis, as shown in FIG. 4. Besides the HBSS equilibrium solution used in the experiment, other equilibrium solutions with ion buffering effect can also complete the loading of DNA, such as PBS buffer solution and the like; the loading system is not limited to 1mg DNA/50OD GHOST/100. Mu.L HBSS buffer used in the experiment, and DNA loading can be accomplished with different DNA/GHOST concentration ratios.

Example 2 validation of the efficiency of macrophage specific transfection with GHOST loaded with GFP eukaryotic expression plasmid

To verify the transfection efficiency of E.coli GHOST into macrophages, the GFP-expressing eukaryotic expression plasmid pMAX-GPF was loaded as described in example 1 to prepare GFP-GHOST complexes, and each was transfected in vitro and in vivo. In the case of in vitro transfection of cells, it is recommended that the transfection be carried out in complete medium at a ratio of 500 GHSOT/cell. After 2 hours of transfection, the E.coli GHOST that had not been taken up was washed away. First we transfected mouse macrophage cell line RAW264.7 and colon cancer cell line CT26 cells with GFP-GHOST and plasmid-unloaded, empty GHOST. As shown in FIG. 5, no GFP expression was detected in CT26 cells, whereas GFP-GHOST transfected RAW264.7 cells had extensive GFP expression. In addition, GFP-GHOST was also able to transfect mouse primary cells efficiently, as shown in FIG. 6.

Example 3 construction of eukaryotic expression plasmid of shRNA targeting Lamp2a and GHOST-shRNA expression vector Complex targeting tumor-associated macrophage

shRNA template sequences were designed against subtype-a specific exon 9 sequences of mouse Lamp2a (NM _ 001017959.2) as shown in table 1. And synthesizing the template sequences into corresponding complementary Oligo DNAs respectively, namely: forward direction: CACC-sense-CGAA-antisense; and (3) reversing: AAAA-sense-TTCG-antisense.

TABLE 1 mouse Lamp2a shRNA template sequences

After annealing to form double-stranded DNA of hairpin structure of each pair of Oligo DNAs, each pair of Oligo DNAs is connected with linear shRNA eukaryotic expression plasmid pENTR/U6 (provided by Invitrogen), inserted into CACC-TTTT site of plasmid pENTR/U6 and recombined into shRNA eukaryotic expression plasmid (sh-L2 a) of target Lamp2 a.

The method described in example 1 is used to load the eukaryotic expression plasmid (sh-L2 a) of shRNA targeting Lamp2a into Escherichia coli GHOST, and GHOST-shRNA expression vector complex targeting tumor-associated macrophages is obtained. It was determined that every 1OD of E.coli GHOST was loaded with approximately 10. Mu.g of DNA, i.e.approximately 1 E.coli GHOST could carry 6000 eukaryotic expression plasmids (sh-L2 a).

Besides the commercial shRNA eukaryotic expression plasmid pENTR/U6 produced by Invitrogen company used in the experiment, other shRNA eukaryotic expression plasmids commonly used in the field, such as pLKO.1, pGPU6, pGPH1 and pGenesil-2, can also be used for constructing shRNA plasmids targeting Lamp2a and loaded into escherichia coli GHOST.

Example 4 Gene interference efficiency verification of E.coli GHOST loaded with Lamp2a shRNA eukaryotic expression plasmid

And (3) transfecting primary mouse macrophages by using escherichia coli GHOST loaded with the Lamp2a shRNA eukaryotic expression plasmid in vitro, and verifying the gene interference efficiency of the system.

First, tumor cell culture supernatant was co-cultured with isolated mouse primary bone marrow-derived macrophages (BMDM) in vitro for 72 hours to activate LAMP2a expression, followed by transfection of these cells with e.coli GHOST (sh-L2 a) loaded with LAMP2a shRNA eukaryotic expression plasmid. As shown in the result of figure 7, after 72 hours of transfection, three shRNAs can effectively inhibit the LAMP2a expression in mouse macrophages, wherein the shRNA expression plasmid (shown in SEQ ID NO: 7) loaded with a No.2 template sequence (SEQ ID NO: 2: CTGCAATCTGATTGATTA) has the most remarkable effect, so that the shRNA expression plasmid loaded with the No.2 template sequence is selected for subsequent experiments.

Next, GHOST (sh-NC group) loaded with shRNA eukaryotic expression plasmid without specific targeting was supplemented as a control to exclude nonspecific effects of E.coli GHOST itself or shRNA expression plasmid on LAMP2a expression in macrophages. The results are shown in FIG. 8, and show that although the sh-NC group has a certain influence on the expression of Lamp2a mRNA, the sh-L2a group significantly inhibits the LAMP2a expression level in macrophages at both protein level and mRNA level.

The results show that the Lamp2a shRNA expression plasmid delivered by the escherichia coli GHOST can effectively target macrophages and inhibit the expression of LAMP2 a.

Example 5 in vivo experiments with E.coli GHOST loaded with Lamp2a shRNA eukaryotic expression plasmid

The expression of LAMP2a in mouse tumor-related macrophages is specifically interfered in vivo by an escherichia coli GHOST-shRNA system, and the gene interference efficiency and the tumor treatment effect of the system are verified.

First, to determine the dose of E.coli GHOST injected in vivo, balb/c mice were injected with 1OD, 2OD, 5OD GHOST or saline (NS) as controls in the tail vein, respectively. After 12 hours, 24 hours and 48 hours, the mice were sacrificed, and the peripheral blood concentrations of IL-1. Beta., IL-6 and TNF-. Alpha.were measured by ELISA, and the results are shown in FIG. 9. Each mouse was selected for intravenous injection of 2OD E.coli GHOST.

In order to examine the interfering effect of the E.coli GHOST-shRNA system in mice, a spontaneous breast cancer mouse model PyMT, a colon cancer cell line CT26 and a mouse breast cancer cell line 4T1 subcutaneous inoculation model were used. For PyMT model, about 4 days after the mouse has the touchable tumor, when the tumor of the experimental mouse reaches approximately the same volume, the mouse is injected with Escherichia coli GHOST which is resuspended in 100 μ L of shRNA eukaryotic expression plasmid (sh-NC group) without specific targeting or shRNA eukaryotic expression plasmid (sh-L2 a group) targeting Lamp2a respectively, or normal saline with the same volume as the control (NS group). Each mouse was injected with 2OD E.coli GHOST every 3 days. Results as shown in fig. 10, sh-L2a treatment significantly inhibited tumor growth in PyMT mice, whereas tumor growth was not significantly different between the NS and sh-NC groups. In the sh-L2 a-treated group, approximately 1/3 of the mice exhibited complete tumor remission. Meanwhile, the expression of LAMP2a in tumor-associated macrophages is also obviously inhibited, as shown in FIG. 11. In addition, as shown in fig. 12, the LAMP2a expression level in other tumor infiltrating immune cells has no obvious change, which indicates that only tumor-associated macrophages are interfered by shRNA delivered by GHOST, and have better targeting.

For the CT26 and 4T1 mouse models, one intratumorally injected control group (i.t.) was added to each of the three treatment groups, except for tail vein injection (i.v.). The dose and frequency of administration was the same as for PyMT mice described above. The results are shown in FIG. 13, the E.coli GHOST-shRNA transfection system also significantly inhibited tumor growth in CT26 and 4T1 mouse models. Similarly, as shown in FIG. 14, LAMP2a expression in tumor-associated macrophages was also significantly inhibited by sh-L2a delivered by E.coli GHOST. The result of the embodiment shows that the GHOST-shRNA compound is used for targeting and interfering the expression of LAMP2a in the tumor-associated macrophages, so that the tumor growth can be effectively inhibited.

As the infiltration degree of macrophages in various solid tumors is obviously related to poor prognosis, such as breast cancer, prostatic cancer, endometrial cancer, bladder cancer, esophageal epithelial cancer, squamous cell carcinoma and the like. Considering the extensive expression of LAMP2a in tumor-associated macrophages, while the degree of infiltration of LAMP2 a-positive macrophages was also correlated with patient survival, as shown in fig. 15, there was a clear manifestation in 145 breast cancer patients. Furthermore, fig. 15 also shows that the expression level of LAMP2a in tumor cells is not significantly correlated with poor prognosis. Meanwhile, since the 9 th exon sequence of Lamp2a is also a highly conserved sequence, for example, the similarity between the mouse and human sequence reaches 91%, shRNA template sequences corresponding to three mouse shRNA template sequences in table 1 and aiming at corresponding sites of human Lamp2a are provided, as shown in table 2, the sequences are also highly similar to the corresponding sequences in table one.

TABLE 2 human shRNA template sequences corresponding to mouse Lamp2a shRNA template sequences

Therefore, the LAMP2a expression in the macrophage related to the specific target interference tumor has high clinical transformation potential, and the GHOST-shRNA expression vector compound has good application prospect in treating macrophage high-infiltration type tumor.

Sequence listing

<110> Sichuan university

<120> GHOST-shRNA expression vector compound targeting tumor-associated macrophages and application thereof

<130> A190052K

<150> 201910005062.4

<151> 2019-01-03

<160> 7

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gactgcagtg cagatgacg 19

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ctgcaacctg attgatta 18

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taaaatatgt agttgtgtc 19

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ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga 60

taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga 120

gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca 180

cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaata cgcgtaccgc 240

tagccaggaa gagtttgtag aaacgcaaaa aggccatccg tcaggatggc cttctgctta 300

gtttgatgcc tggcagttta tggcgggcgt cctgcccgcc accctccggg ccgttgcttc 360

acaacgttca aatccgctcc cggcggattt gtcctactca ggagagcgtt caccgacaaa 420

caacagataa aacgaaaggc ccagtcttcc gactgagcct ttcgttttat ttgatgcctg 480

gcagttccct actctcgcgt taacgctagc atggatgttt tcccagtcac gacgttgtaa 540

aacgacggcc agtcttaagc tcgggcccca aataatgatt ttattttgac tgatagtgac 600

ctgttcgttg caacaaattg atgagcaatg cttttttata atgccaactt tgtacaaaaa 660

agcaggcttt aaaggaacca attcagtcga ctggatccgg taccaaggtc gggcaggaag 720

agggcctatt tcccatgatt ccttcatatt tgcatatacg atacaaggct gttagagaga 780

taattagaat taatttgact gtaaacacaa agatattagt acaaaatacg tgacgtagaa 840

agtaataatt tcttgggtag tttgcagttt taaaattatg ttttaaaatg gactatcata 900

tgcttaccgt aacttgaaag tatttcgatt tcttggcttt atatatcttg tggaaaggac 960

gaaacaccgc tgcaatctga ttgattacga ataatcaatc agattgcagt tttttctaga 1020

cccagctttc ttgtacaaag ttggcattat aagaaagcat tgcttatcaa tttgttgcaa 1080

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ttacataaac agtaatacaa ggggtgttat gagccatatt caacgggaaa cgtcgaggcc 1320

gcgattaaat tccaacatgg atgctgattt atatgggtat aaatgggctc gcgataatgt 1380

cgggcaatca ggtgcgacaa tctatcgctt gtatgggaag cccgatgcgc cagagttgtt 1440

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ctggctgacg gaatttatgc ctcttccgac catcaagcat tttatccgta ctcctgatga 1560

tgcatggtta ctcaccactg cgatccccgg aaaaacagca ttccaggtat tagaagaata 1620

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gattcctgtt tgtaattgtc cttttaacag cgatcgcgta tttcgtctcg ctcaggcgca 1740

atcacgaatg aataacggtt tggttgatgc gagtgatttt gatgacgagc gtaatggctg 1800

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ttgtattgat gttggacgag tcggaatcgc agaccgatac caggatcttg ccatcctatg 1980

gaactgcctc ggtgagtttt ctccttcatt acagaaacgg ctttttcaaa aatatggtat 2040

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aagctcatga ccaaaatccc ttaacgtgag ttacgcgtcg ttccactgag cgtcagaccc 2220

cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt 2280

gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac 2340

tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg tccttctagt 2400

gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct 2460

gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga 2520

ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac 2580

acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagcattg 2640

agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt 2700

cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc 2760

tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg 2820

gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc 2880

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Claims (14)

1. The GHOST-shRNA expression vector compound of the targeted tumor-related macrophage is characterized in that the GHOST-shRNA compound is bacterial GHOST loaded with shRNA eukaryotic expression vector for inhibiting LAMP2a expression; the shRNA eukaryotic expression vector for inhibiting LAMP2a expression is a shRNA sequence capable of expressing a 9 th exon sequence aiming at Lamp2 a; the shRNA template sequence in the shRNA eukaryotic expression vector for inhibiting LAMP2a expression is SEQ ID No.2; the bacterial GHOST is GHOST in escherichia coli.

2. The GHOST-shRNA expression vector complex targeting tumor-associated macrophages of claim 1, wherein: the shRNA eukaryotic expression vector for inhibiting LAMP2a expression is a plasmid vector.

3. The GHOST-shRNA expression vector complex targeting tumor-associated macrophages of claim 2, wherein: the plasmid vector is at least one of pENTR/U6, pLKO.1, pGPU6, pGPH1 or pGenesil-2.

4. The tumor associated macrophage targeting GHOST-shRNA expression vector complex of claim 1, wherein: the bacterial GHOST is GHOST of Escherichia coli DH5 alpha.

5. The tumor associated macrophage targeting GHOST-shRNA expression vector complex of claim 1, wherein: the nucleotide sequence of the shRNA eukaryotic expression vector for inhibiting LAMP2a expression is shown in SEQ ID No. 7.

6. The GHOST-shRNA expression vector composition targeting tumor-associated macrophages of claim 1An article, characterized in that: the proportion between the bacterial GHOST and the shRNA eukaryotic expression vector for inhibiting LAMP2a expression is as follows: every 10 th ⁸ The escherichia coli GHOST is loaded with 0.3-10 mu g shRNA eukaryotic expression vector.

7. The GHOST-shRNA expression vector complex targeting tumor-associated macrophages of claim 6, wherein: the proportion of the bacterial GHOST to the shRNA eukaryotic expression vector for inhibiting LAMP2a expression is as follows: every 10 th ⁸ Each escherichia coli GHOST loads 1-5 mu g shRNA eukaryotic expression vector.

8. The tumor associated macrophage targeting GHOST-shRNA expression vector complex of claim 1, wherein: the shRNA eukaryotic expression vector for inhibiting LAMP2a expression is loaded in the bacterial GHOST through the potential difference between the inside and the outside of the bacterial GHOST.

9. Use of the GHOST-shRNA expression vector complex targeting tumor-associated macrophages according to any one of claims 1 to 8 in the preparation of a medicament for treating macrophage highly invasive tumor.

10. Use according to claim 9, characterized in that: the macrophage high-infiltration type tumor is at least one of breast cancer, prostate cancer, endometrial cancer, bladder cancer, kidney cancer, follicular lymphoma, malignant uveal melanoma, esophageal epithelial cancer or squamous cell carcinoma.

11. The medicine for treating macrophage high-infiltration type tumor is characterized in that: the preparation is prepared by adding pharmaceutically acceptable auxiliary materials or auxiliary components into the GHOST-shRNA expression vector compound of the targeted tumor-associated macrophage as claimed in any one of claims 1-8.

12. The medicament for treating macrophage highly-invasive tumor according to claim 11, wherein: the macrophage high-infiltration tumor is at least one of breast cancer, prostate cancer, endometrial cancer, bladder cancer, kidney cancer, follicular lymphoma, malignant uveal melanoma, esophageal epithelial cancer or squamous cell carcinoma.

13. The medicament for treating macrophage highly-invasive tumor according to claim 11 or 12, wherein: the preparation is an injection.

14. The method for preparing the GHOST-shRNA expression vector complex targeting tumor-associated macrophages according to any one of claims 1 to 8, comprising the steps of:

a. designing an shRNA sequence aiming at Lamp2a, and constructing an expression vector capable of expressing the shRNA sequence;

b. and loading the constructed shRNA eukaryotic expression plasmid into bacterial GHOST to prepare the GHOST-shRNA expression vector compound.

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