A CRISPR / Cas9 nanosystem

Abstract

The present invention relates to a CRISPR / Cas9 nanosystem. Specifically, the present invention provides a CRISPR / Cas9 nanosystem, and the CRISPR / Cas9 nanosystem includes a carrier, a sonosensitizer, and a CRISPR / Cas9 system; the carrier is loaded with the sonosensitizer and the CRISPR / Cas9 system. The carrier of the present invention is loaded with the sonosensitizer and the CRISPR / Cas9 system, and the CRISPR / Cas9 nanosystem has excellent lysosomal escape ability under ultrasonic irradiation conditions, thereby avoiding degradation by lysosomes and enhancing gene editing efficiency.

Description

Technical Field

[0001] The present invention relates to the biological field, and in particular, to a CRISPR / Cas9 nanosystem. Background Art

[0002] CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) / Cas9, an emerging genome editing technology, boasts advantages such as simple design, strong specificity, and high efficiency. It has brought breakthroughs in the regulation and application of targeted genome editing and holds broad promise in biomedicine. However, addressing biosafety concerns and achieving efficient in vivo delivery of protein / nucleic acid complexes while minimizing off-target effects remain key scientific challenges facing current CRISPR / Cas9 delivery systems.

[0003] The CRISPR / Cas9 system is characterized by its large size and easy degradation. Without the assistance of a corresponding delivery vehicle, it is almost impossible for it to enter mammalian cells, whether in the form of DNA, mRNA, or protein. Although viral vectors have high delivery efficiency, they have biosafety issues, which hinder their widespread application. In recent years, various non-viral delivery methods have been reported, but they are not suitable for in vivo application and systemic use, or lack nuclear targeting. These vectors can deliver Cas9 protein and sgRNA to the target area, but they can also transfer gene editing materials.

[0004] Currently, there are many problems in the design of CRISPR / Cas9 delivery systems, including the design of sgRNA, the selection and design of carrier materials, and the design of stimulus-responsive switches. Therefore, it is necessary to design efficient sgRNA and target its delivery to tumor cells. In terms of delivery vectors, CRISPR / Cas9 delivery system vectors are currently mainly based on viral vectors, but viral vectors have certain safety issues. At the same time, in the design of stimulus-responsive switches, after the CRISPR / Cas9 system effectively enters the cell, how to avoid cytoplasmic degradation, how to achieve lysosomal escape, and how to efficiently release the corresponding Cas9 / sgRNA complex and efficiently enter the nucleus are the key to determining whether the target gene can be effectively knocked out.

[0005] Therefore, there is a need in the art to develop a CRISPR / Cas9 nanosystem for efficient delivery. Summary of the Invention

[0006] The purpose of the present invention is to provide a CRISPR / Cas9 nanosystem with high safety and efficient delivery.

[0007] To achieve the above objectives, the technical solutions adopted in this application are:

[0008] In the first aspect of the present invention, a CRISPR / Cas9 nanosystem is provided, and the CRISPR / Cas9 nanosystem includes a carrier, a photosensitizer, and a CRISPR / Cas9 system;

[0009] The carrier loads the photosensitizer and the CRISPR / Cas9 system.

[0010] Preferably, the photosensitizer includes hematoporphyrin monomethyl ether.

[0011] In the second aspect of the present invention, a method for preparing the CRISPR / Cas9 nanosystem as described in the first aspect of the present invention is provided, and the method includes:

[0012] (1) Mix and stir the photosensitizer, 2-methylimidazole, and zinc nitrate, centrifuge, and wash to obtain MH;

[0013] (2) Incubate MH and the CRISPR / Cas9 system to obtain the CRISPR / Cas9 nanosystem.

[0014] Preferably, the mass ratio of MH to the CRISPR / Cas9 system is 3 - 5:1, more preferably 3.5 - 4.5:1.

[0015] Preferably, the method includes the steps:

[0016] (1) Dissolve 1.8 - 2.0 g of 2-methylimidazole and 1.2 - 1.4 g of zinc nitrate in 18 - 22 ml of methanol respectively to obtain a 2-methylimidazole solution and a zinc nitrate solution.

[0017] (2) Under mechanical stirring at room temperature, add hematoporphyrin monomethyl ether (190 - 210 μL, 1.8 - 2.2 mg / mL) to the 2-methylimidazole solution, then dropwise add the zinc nitrate solution, stir at room temperature, centrifuge, and wash to obtain MH.

[0018] (3) Incubate MH and the CRISPR / Cas9 system to obtain the CRISPR / Cas9 nanosystem.

[0019] In the third aspect of the present invention, a probiotic CRISPR / Cas9 nanosystem is provided, and the probiotic CRISPR / Cas9 nanosystem includes lactic acid bacteria and the CRISPR / Cas9 nanosystem as described in the first aspect of the present invention.

[0020] Preferably, the probiotic CRISPR / Cas9 nanosystem includes a self-driven probiotic CRISPR / Cas9 nanosystem.

[0021] Preferably, the lactic acid bacteria load the CRISPR / Cas9 nanosystem.

[0022] Preferably, the lactic acid bacteria include Lactobacillus rhamnosus.

[0023] The fourth aspect of the present invention provides a method for preparing the probiotic CRISPR / Cas9 nanosystem as described in the third aspect of the present invention, the method comprising:

[0024] (a) The CRISPR / Cas9 nanosystem is mixed with lactic acid bacteria to obtain a probiotic CRISPR / Cas9 nanosystem.

[0025] Preferably, the number of the lactic acid bacteria is 0.5×107 CFU to 1.5×107 CFU, more preferably 0.8×107 CFU to 1.2×107 CFU.

[0026] Preferably, the mass of the CRISPR / Cas9 nanosystem is 0.8-1.2 mg.

[0027] Preferably, the stirring temperature is room temperature.

[0028] The fifth aspect of the present invention provides a use of the CRISPR / Cas9 nanosystem as described in the first aspect of the present invention or the probiotic CRISPR / Cas9 nanosystem as described in the third aspect of the present invention, for preparing a composition, wherein the composition is used for gene editing.

[0029] Preferably, the composition is a pharmaceutical composition or a reagent composition

[0030] Preferably, the composition further comprises a pharmaceutically and reagent-acceptable carrier.

[0031] Preferably, the composition is in the form of a solid preparation, a liquid preparation or a semisolid preparation.

[0032] Preferably, the composition is in the form of an oral preparation, an external preparation or an injection preparation.

[0033] Preferably, the composition is in the form of tablets, injections, infusions, ointments, gels, solutions, microspheres or films.

[0034] Preferably, the injection preparation is an intravenous injection preparation.

[0035] Preferably, after administering the CRISPR / Cas9 nanosystem as described in the first aspect of the present invention or the probiotic CRISPR / Cas9 nanosystem as described in the third aspect of the present invention, the target site for gene editing (such as target cells, target organs) is subjected to ultrasonic irradiation treatment.

[0036] The sixth aspect of the present invention provides a composition, which comprises the CRISPR / Cas9 nanosystem as described in the first aspect of the present invention or the probiotic CRISPR / Cas9 nanosystem as described in the third aspect of the present invention.

[0037] Preferably, the composition is a pharmaceutical composition or a reagent composition.

[0038] Preferably, the composition further comprises a pharmaceutically or reagent-acceptable carrier.

[0039] Preferably, the dosage form of the composition is a solid preparation, a liquid preparation or a semi-solid preparation.

[0040] Preferably, the dosage form of the composition is an oral preparation, a topical preparation or an injection preparation.

[0041] Preferably, the dosage form of the composition is a tablet, an injection, an infusion, an ointment, a gel, a solution, a microsphere or a film.

[0042] Preferably, the injection preparation is an intravenous injection preparation.

[0043] The seventh aspect of the present invention provides a method for gene editing of cells, which comprises contacting the CRISPR / Cas9 nanosystem as described in the first aspect of the present invention or the probiotic CRISPR / Cas9 nanosystem as described in the third aspect of the present invention with cells, so as to perform gene editing on the cells.

[0044] Preferably, the method is an in vitro method.

[0045] Preferably, the method is a non-diagnostic and non-therapeutic method.

[0046] Preferably, the contact is an in vitro contact.

[0047] Preferably, the contact includes contact under ultrasonic irradiation.

[0048] Preferably, after administering the CRISPR / Cas9 nanosystem as described in the first aspect of the present invention or the probiotic CRISPR / Cas9 nanosystem as described in the third aspect of the present invention, the cells are subjected to ultrasonic irradiation treatment.

[0049] The eighth aspect of the present invention provides a method for gene editing, which comprises: administering the CRISPR / Cas9 nanosystem as described in the first aspect of the present invention or the probiotic CRISPR / Cas9 nanosystem as described in the third aspect of the present invention to a desired subject, so as to perform gene editing.

[0050] Preferably, the subject includes a human or a non-human mammal.

[0051] Preferably, the non-human mammals include cows, horses, sheep, dogs, cats or mice.

[0052] Preferably, the administration is oral administration or injection administration.

[0053] Preferably, the injection administration is intravenous injection administration.

[0054] Preferably, the target site of gene editing (such as target cells, target organs) is treated by ultrasonic irradiation.

[0055] Preferably, after administering the CRISPR / Cas9 nanosystem as described in the first aspect of the present invention or the probiotic CRISPR / Cas9 nanosystem as described in the third aspect of the present invention, the target site of gene editing (such as target cells, target organs) is treated by ultrasonic irradiation.

[0056] The ninth aspect of the present invention provides a use of an ultrasonic apparatus for preparing a device, and the device is used for one or more uses selected from the following group:

[0057] (i) improving the retention and / or degradation of the CRISPR / Cas9 nanosystem as described in the first aspect of the present invention or the probiotic CRISPR / Cas9 nanosystem as described in the third aspect of the present invention by lysosomes of cells through ultrasonic irradiation; and / or

[0058] (ii) improving the gene editing efficiency of the CRISPR / Cas9 nanosystem as described in the first aspect of the present invention or the probiotic CRISPR / Cas9 nanosystem as described in the third aspect of the present invention by ultrasonic irradiation.

[0059] Preferably, the improvement includes reducing, overcoming or avoiding.

[0060] Preferably, after administering the CRISPR / Cas9 nanosystem as described in the first aspect of the present invention or the probiotic CRISPR / Cas9 nanosystem as described in the third aspect of the present invention, the target site of gene editing (such as target cells, target organs) is treated by ultrasonic irradiation.

[0061] The tenth aspect of the present invention provides a device for gene editing, and the device includes the CRISPR / Cas9 nanosystem as described in the first aspect of the present invention or the probiotic CRISPR / Cas9 nanosystem as described in the third aspect of the present invention; and an ultrasonic device.

[0062] Preferably, the system or device further includes an instruction manual or a label, and the instruction manual or the label records:

[0063] After administering the CRISPR / Cas9 nanosystem as described in the first aspect of the present invention or the probiotic CRISPR / Cas9 nanosystem as described in the third aspect of the present invention to the desired object, ultrasonic irradiation treatment is performed on the target site of gene editing (such as target cells, target organs).

[0064] Within the scope of the present invention, the above technical features of the present invention and the technical features specifically described hereinafter can be combined with each other to form new or preferred technical solutions. Brief Description of the Drawings

[0065] Figure 1 For the preparation of the self-driven probiotic CRISPR / Cas9 nanosystem, TEM detection.

[0066] Figure 2 For in vivo imaging to verify the targeting ability of the self-driven probiotic CRISPR / Cas9 nanosystem.

[0067] Figure 3 For CLSM to observe the subcellular distribution of the MHS nanosystem after incubation with or without ultrasonic irradiation for 1 or 3 hours, where US is ultrasonic irradiation treatment.

[0068] Figure 4 For NGS sequencing to detect the mutation rate of the target gene IDO1. Ultrasonic can significantly improve the gene editing efficiency of the MHS nanosystem, where US is ultrasonic irradiation treatment.

[0069] Figure 5 For CLSM to detect the expression of the target protein IDO. Ultrasonic can significantly improve the gene editing efficiency of the MHS nanosystem and reduce the expression level of the protein IDO. Where US is ultrasonic irradiation treatment.

[0070] Figure 6 For Western blot and CLSM to detect the effect of triggering tumor immunogenic cell death of the system under ultrasonic action. (a) Specific protein expression after Western blot analysis of DAMPs (HMGB1, CRT and HSP70). (b-d) Specific protein expression after immunofluorescence analysis of DAMPs, including HMGB1, CRT and HSP70, where US is ultrasonic irradiation treatment.

[0071] Figure 7Flow cytometry and ELSA analysis of the in vitro immune activation effect. (a) Schematic diagram of the in vitro DC maturation experiment. In the upper chamber, 4T1 cells were treated with US alone and incubated with MH, MH + US, MHS, and MHS + US. BMDCs were cultured in the lower chamber. After 24 hours of coculture, BMDCs were harvested for analysis. (b) BMDC supernatant secreted IL-12p70 and IL-2. (c, d) Representative flow cytometric images and statistical data of mature BMDCs (CD80+CD86+CD11c+) after treatment in each group. US represents ultrasound irradiation.

[0072] Figure 8 Transcriptome sequencing revealed that LGG can activate multiple immune-related signaling pathways. (a, b) Volcano plots and heat maps of gene expression changes before and after LGG treatment (P < 0.05, |foldchange| ≥ 2). (c) KEGG analysis of differential gene expression profiles after LGG treatment.

[0073] Figure 9 Figure 3. The effects of LGG-MHS+US on 4T1 tumors in vivo. (a) Schematic diagram of the in vivo primary tumor treatment process. (b) 4T1 tumor growth curves after treatment with PBS, LGG, MHS, LGG-MHS, MHS+US, LGG-MH+US, LGG-MHI+US, and LGG-MHS+US (n=5). To compare the efficacy of traditional IDO small molecule inhibitors with IDO1 gene knockout, the IDO inhibitor NLG919 was added as a CRISPR control. I refers to an IDO inhibitor. (c) Average tumor growth curves for each group (n=5). US represents ultrasound irradiation.

[0074] Figure 10 It is a flow cytometer for effector memory T cells, where US is ultrasound irradiation treatment. DETAILED DESCRIPTION

[0075] The present invention has developed a CRISPR / Cas9 nanosystem, which includes a vector, a sonosensitizer, and a CRISPR / Cas9 system. The vector carries the sonosensitizer and the CRISPR / Cas9 system. The CRISPR / Cas9 nanosystem has excellent lysosomal escape ability under ultrasound irradiation, thereby avoiding lysosomal degradation and enhancing gene editing efficiency. The present invention also developed a non-viral lactic acid bacteria-loaded CRISPR / Cas9 nanosystem for gene editing, which is highly safe and can exert excellent tumor targeting and anti-tumor effects.

[0076] the term

[0077] As used herein, the terms "comprising", "including" and "containing" are used interchangeably and include not only open-ended definitions but also semi-closed and closed definitions. In other words, the terms include "consisting of" and "consisting essentially of".

[0078] CRISPR / Cas9 Nanosystem and Its Preparation Method

[0079] The present invention develops a CRISPR / Cas9 nanosystem, which comprises a carrier, a photosensitizer and a CRISPR / Cas9 system.

[0080] The carrier loads the photosensitizer and the CRISPR / Cas9 system.

[0081] In a preferred embodiment of the present invention, the photosensitizer includes (but is not limited to) hematoporphyrin monomethyl ether.

[0082] The present invention also provides a method for preparing the CRISPR / Cas9 nanosystem of the present invention, and the method comprises:

[0083] (1) Mix and stir the photosensitizer, 2-methylimidazole and zinc nitrate, centrifuge and wash to obtain MH.

[0084] (2) Incubate MH and the CRISPR / Cas9 system to obtain the CRISPR / Cas9 nanosystem.

[0085] In a preferred embodiment of the present invention, the mass ratio of MH to the CRISPR / Cas9 system is 3 - 5:1, preferably 3.5 - 4.5:1.

[0086] Typically, the method comprises the steps of:

[0087] (1) Dissolve 1.8 - 2.0 g of 2-methylimidazole and 1.2 - 1.4 g of zinc nitrate in 18 - 22 ml of methanol respectively to obtain a 2-methylimidazole solution and a zinc nitrate solution.

[0088] (2) Under mechanical stirring at room temperature, add hematoporphyrin monomethyl ether (190 - 210 μL, 1.8 - 2.2 mg / mL) to the 2-methylimidazole solution, then dropwise add the zinc nitrate solution, stir at room temperature, centrifuge and wash to obtain MH.

[0089] (3) Incubate MH and the CRISPR / Cas9 system to obtain the CRISPR / Cas9 nanosystem.

[0090] Probiotic CRISPR / Cas9 Nanosystem

[0091] The present invention provides a probiotic CRISPR / Cas9 nanosystem, and the probiotic CRISPR / Cas9 nanosystem includes lactic acid bacteria and the CRISPR / Cas9 nanosystem of the present invention.

[0092] In a preferred embodiment of the present invention, the probiotic CRISPR / Cas9 nanosystem includes a self-driven probiotic CRISPR / Cas9 nanosystem.

[0093] In a preferred embodiment of the present invention, the lactic acid bacteria are loaded with the CRISPR / Cas9 nanosystem.

[0094] Preferably, the lactic acid bacteria include Lactobacillus rhamnosus.

[0095] The present invention also provides a method for preparing the probiotic CRISPR / Cas9 nanosystem of the present invention, and the method includes:

[0096] (a) Mixing and stirring the CRISPR / Cas9 nanosystem with lactic acid bacteria to obtain the probiotic CRISPR / Cas9 nanosystem.

[0097] Preferably, the number of the lactic acid bacteria is 0.5×107 CFU to 1.5×107 CFU, more preferably 0.8×107 CFU to 1.2×107 CFU.

[0098] Preferably, the mass of the CRISPR / Cas9 nanosystem is 0.8 - 1.2 mg.

[0099] Preferably, the stirring temperature is room temperature.

[0100] Use

[0101] The present invention provides a use of the CRISPR / Cas9 nanosystem of the present invention or the probiotic CRISPR / Cas9 nanosystem of the present invention for preparing a composition, and the composition is used for gene editing.

[0102] Preferably, after administering the CRISPR / Cas9 nanosystem as described in the first aspect of the present invention or the probiotic CRISPR / Cas9 nanosystem as described in the third aspect of the present invention, ultrasonic irradiation treatment is performed on the target site (such as target cells, target organs) for gene editing.

[0103] The present invention provides a use of an ultrasonic instrument for preparing a device, and the device is used for one or more uses selected from the following group:

[0104] (i) improving the retention and / or degradation of the CRISPR / Cas9 nanosystem of the present invention or the probiotic CRISPR / Cas9 nanosystem of the present invention by lysosomes by ultrasound irradiation; and / or

[0105] (ii) Improving the gene editing efficiency of the CRISPR / Cas9 nanosystem of the present invention or the probiotic CRISPR / Cas9 nanosystem of the present invention by ultrasonic irradiation.

[0106] Preferably, said improvement includes reducing, overcoming or avoiding.

[0107] Preferably, after administering the CRISPR / Cas9 nanosystem of the present invention or the probiotic CRISPR / Cas9 nanosystem of the present invention, the target site for gene editing (such as target cells, target organs) is subjected to ultrasonic irradiation treatment.

[0108] method

[0109] The present invention provides a method for gene editing of cells, comprising: contacting the CRISPR / Cas9 nanosystem described in the present invention or the probiotic CRISPR / Cas9 nanosystem described in the present invention with cells, thereby gene editing the cells.

[0110] In a preferred embodiment of the present invention, the method is an in vitro method.

[0111] In a preferred embodiment of the present invention, the method is a non-diagnostic and non-therapeutic method.

[0112] In a preferred embodiment of the present invention, the contact is in vitro contact.

[0113] In a preferred embodiment of the present invention, the contacting includes contacting under ultrasonic irradiation.

[0114] Preferably, after administering the CRISPR / Cas9 nanosystem as described in the first aspect of the present invention or the probiotic CRISPR / Cas9 nanosystem as described in the third aspect of the present invention, the cells are subjected to ultrasonic irradiation treatment.

[0115] The present invention also provides a method for gene editing, which comprises administering the CRISPR / Cas9 nanosystem of the present invention or the probiotic CRISPR / Cas9 nanosystem of the present invention to a desired subject, thereby performing gene editing.

[0116] In a preferred embodiment of the present invention, the subject includes humans or non-human mammals.

[0117] Preferably, the non-human mammals include cows, horses, sheep, dogs, cats or mice.

[0118] In a preferred embodiment of the present invention, the administration is oral administration or injection administration.

[0119] Preferably, the injection administration is intravenous injection administration.

[0120] In a preferred embodiment of the present invention, the target site of gene editing (such as target cells, target organs) is treated with ultrasonic irradiation.

[0121] In a preferred embodiment of the present invention, after administering the CRISPR / Cas9 nanosystem as described in claim 1 or the probiotic CRISPR / Cas9 nanosystem as described in claim 3, the target site of gene editing (such as target cells, target organs) is treated with ultrasonic irradiation.

[0122] Device

[0123] The present invention provides a device for gene editing, and the device includes the CRISPR / Cas9 nanosystem as described in the present invention or the probiotic CRISPR / Cas9 nanosystem as described in the present invention; and an ultrasonic device.

[0124] Preferably, the system or device further includes an instruction manual or a label, and the instruction manual or the label records:

[0125] Administer the CRISPR / Cas9 nanosystem as described in the present invention or the probiotic CRISPR / Cas9 nanosystem as described in the present invention to a desired subject, and treat the target site of gene editing (such as target cells, target organs) with ultrasonic irradiation.

[0126] Composition

[0127] The composition as described in the present invention is preferably a pharmaceutical composition or a reagent composition, and the composition as described in the present invention may include a pharmaceutically or reagent-acceptable carrier.

[0128] As used herein, "pharmaceutically acceptable carrier" refers to one or more compatible solid, semi-solid, liquid or gel fillers, which are suitable for human or animal use, and must have sufficient purity and sufficiently low toxicity. "Compatibility" means that the components in the pharmaceutical composition and the active ingredients and the admixture between them do not significantly reduce the drug efficacy.

[0129] It should be understood that in the present invention, the pharmaceutically acceptable carrier is not particularly limited, and materials commonly used in the art can be selected, or prepared by conventional methods, or obtained from the market. Some examples of pharmaceutically acceptable carriers include cellulose and its derivatives (such as methylcellulose, ethylcellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose, etc.), gelatin, talc, solid lubricants (such as stearic acid, magnesium stearate), calcium sulfate, vegetable oils (such as soybean oil, sesame oil, peanut oil, olive oil, etc.), polyols (such as propylene glycol, glycerol, mannitol, sorbitol, etc.), emulsifiers (such as Tween), wetting agents (such as sodium lauryl sulfate), buffering agents, chelating agents, thickening agents, pH regulators, transdermal penetration enhancers, coloring agents, flavoring agents, stabilizers, antioxidants, preservatives, bacteriostatic agents, pyrogen-free water, etc.

[0130] In a preferred embodiment of the present invention, the dosage form of the composition is a solid preparation, a liquid preparation or a semi-solid preparation.

[0131] In a preferred embodiment of the present invention, the dosage form of the composition is an oral preparation, a topical preparation or an injection preparation

[0132] Preferably, the dosage form of the composition is a tablet, an injection, an infusion, an ointment, a gel, a solution, a microsphere or a film.

[0133] Preferably, the injection preparation is an intravenous injection preparation.

[0134] The pharmaceutical preparation should be compatible with the administration route. The drugs of the present invention can also be used together with other co-therapeutic agents (including before, during or after use). When using the pharmaceutical composition or preparation, a safe and effective amount of the drug is administered to the desired subject (such as a human or non-human mammal), and the safe and effective amount is usually at least about 10 micrograms / kg body weight, and in most cases does not exceed about 8 mg / kg body weight. Preferably, the dose is about 10 micrograms / kg body weight - about 1 mg / kg body weight. Of course, the specific dose should also consider factors such as the administration route and the health status of the patient, which are within the scope of the skills of a skilled physician.

[0135] The main excellent technical effects of the present invention include:

[0136] 1. The present invention develops a CRISPR / Cas9 nanosystem, which has excellent lysosomal escape ability under ultrasonic irradiation conditions, thereby avoiding degradation by lysosomes and enhancing gene editing efficiency.

[0137] 2. The present invention develops a non-viral vector lactic acid bacteria loaded with CRISPR / Cas9 nanosystem for gene editing, which has high safety and can exhibit excellent tumor targeting and anti-tumor effects.

[0138] The present invention will be further described below in conjunction with specific embodiments. It should be understood that the following specific embodiments are based on the present technical solution and give detailed implementation manners and specific operation processes, but the protection scope of the present invention is not limited to this embodiment.

[0139] Example 1

[0140] 1. Preparation of self-driven probiotic CRISPR / Cas9 nanosystem (LGG-MHS)

[0141] (1) 2-methylimidazole (1.910 g) and zinc nitrate (1.314 g) were respectively dissolved in methanol (20 mL) to obtain a 2-methylimidazole solution and a zinc nitrate solution.

[0142] (2) Under mechanical stirring at room temperature, hematoporphyrin monomethyl ether (HMME, 200 μL, 2 mg / mL) was slowly added to the 2-methylimidazole solution. After 10 minutes, the zinc nitrate solution was added dropwise. After stirring at room temperature for 24 hours, it was washed three times with ddH2O and then centrifuged to obtain MH.

[0143] (3) MH and the CRISPR / Cas9 system (the mass ratio of MH to the CRISPR / Cas9 system was 4:1) were incubated at 37 °C, centrifuged, and washed three times with ddH2O to remove residues, obtaining the MHS nanosystem.

[0144] (4) MHS and Lactobacillus rhamnosus (LGG, Lactobacillus genus) (PBS = 1 mL, LGG = 1×107 CFU, MHS = 1 mg) were further stirred in PBS for 24 hours to obtain the self-driven probiotic CRISPR / Cas9 nanosystem (LGG-MHS). The transmission electron microscope (TEM) image of LGG-MHS is as Figure 1 shown.

[0145] 2. Targeting ability of self-driven probiotic CRISPR / Cas9 nanosystem (LGG-MHS)

[0146] Nine 4T1 tumor-bearing mouse models were established and randomly divided into the MHS group, the LGG group, and the LGG-MHS group. When the tumor volume reached about 200 mm3, Cy5.5-labeled MHS (200 μL, Cy5.5-MHS = 10 mg / kg, Cy5.5 = 10 μg / mL), Cy5.5-labeled LGG (200 μL, Cy5.5-LGG = 1×107 CFU, Cy5.5 = 10 mg / kg, Cy5.5 = 10 μg / mL), and Cy5.5-labeled LGG-MHS (200 μL, LGG = 1×107 CFU, Cy5.5-MHS = 10 mg / kg, Cy5.5 = 10 μg / mL) were injected intravenously. Mice were anesthetized at different time points (0, 2, 4, 6, 8, 12, 48, 72 h) and imaged using the VISQUE imaging system.

[0147] Targeting ability of the self-driven probiotic CRISPR / Cas9 nanosystem (LGG-MHS) Figure 2 As shown, from Figure 2 it can be seen that LGG-MHS has excellent tumor targeting ability.

[0148] 3. Examine the control ability of ultrasound on the CRISPR / Cas9 nanosystem

[0149] CLSM (ZEISSLSM900) was used to observe the lysosomal escape of Cy5.5-labeled MHS for recording the intracellular distribution of Cas9 / sgRNA.

[0150] 4T1 cells were pre-seeded into a CLSM-specific culture dish at a density of 1×105 and incubated for 24 hours to adhere to the culture plate. Then, 4T1 cells were incubated with the MHS nanosystem (100 μg / mL) with or without ultrasound irradiation (1.0 MHz, 1.0 W / cm2, 50% duty cycle, 5 minutes) for 1 or 3 hours. DAPI (10 μg / mL) and LysoTracker (0.5 μg / mL) were used to stain the cell nuclei blue and lysosomes green, respectively.

[0151] CLSM was used to observe the subcellular distribution of the MHS nanosystem after incubation with or without ultrasound irradiation for 1 or 3 hours (as Figure 3 shown), and from Figure 3 it can be seen that under ultrasound stimulation, the MHS nanosystem has excellent lysosomal escape ability.

[0152] Meanwhile, the gene editing ability of the target gene was further detected. 4T1 cells were cultured in a 6-well plate for 24 hours (1×105 cells per well), and the medium was replaced with fresh medium without serum. After 1 hour, MH / MHS (100 μg / mL) was added to the wells of the MH, MHS, MH+US, and MHS+US groups. After co-incubation for 12 h, only the US group, MH+US group, and MHS+US group received US (ultrasonic irradiation) (1.0 MHz, 1.0 W / cm2, 50% duty cycle, 5 minutes). The medium was replaced with 2 mL of fresh medium containing 10% FBS. Then, the IDO1 mutant gene groups after different treatments were analyzed. Genomic DNA of the treated 4T1 cells was extracted for PCR analysis (n = 3). Deep sequencing was performed after purification and collection of the PCR products.

[0153] NGS sequencing was used to detect the mutation rate of the target gene IDO1 Figure 4 As shown, from Figure 4 it can be seen that ultrasound can significantly improve the gene editing efficiency of the MHS nanosystem.

[0154] 4. Investigate the immune activation ability after knocking out IDO1 by the CRISPR / Cas9 nanosystem

[0155] CLSM was used to detect the IDO expression. 4T1 cancer cells were seeded into CLSM-specific culture dishes at a density of 1×105 cells / dish and cultured overnight for cell attachment. The cells were stimulated with IFN-γ (1 ng / mL) for 12 hours and subjected to different treatments, including control, US only, MH, MHS, MH+US, and MHS+US (MH / MHS = 100 μg / mL, 1.0 MHz, 1.0 W / cm2, 50% duty cycle, 5 minutes). After washing 3 times with PBS, the cells were incubated with anti-IDO antibody (Cell Signaling Technology 86630) for 1 hour and stained with Alexa Fluor 555-goat anti-rabbit IgG for 1 hour. The cells were stained with DAPI for 20 minutes, and fluorescence images of the cells were captured using CLSM. From Figure 5 it can be seen that the MHS nanosystem can effectively reduce the expression of the protein IDO under ultrasonic irradiation.

[0156] 5. Verify the immune activation ability of ultrasound

[0157] Detection of in vitro immunogenic cell death (ICD). To determine the ICD of tumor cells induced by the CRISPR / Cas9 nanosystem, the surface expression of CRT, the extracellular release of HMGB1 and HSP70 were detected by in vitro immunofluorescence. Generally, 4T1 cells (1×105 cells per well) were seeded in CLSM-specific culture dishes. Then, the cells were incubated with PBS, US only, MH, MHS, MH+US, and MHS+US for 6 hours, and then irradiated (1.0 MHz, 1.0 W / cm, 50% duty cycle, 5 minutes) with or without US irradiation. After further incubation for 12 hours, the cells were washed 3 times with PBS, incubated with rabbit monoclonal antibodies against HMGB1, HSP70, and CRT for 1 hour, and stained with Alexa Fluor 488 or 555 goat anti-rabbit IgG for 1 hour. The cells were stained with DAPI for 20 minutes and observed using CLSM. Bone marrow-derived dendritic cells (BMDCs) were extracted from 6-week-old Balb / c mice to study DC maturation in vitro. 4T1 cells were pretreated with PBS, US only, MH, MHS, MH+US, and MHS+US for 12 hours (MH / MHS = 100 μg / mL, 1.0 MHz, 1.0 W / cm2, 50% duty cycle, 5 minutes). Then, 1×106 immature DC cells were co-cultured with 1×105 pretreated 4T1 cells in a transwell system for 24 hours. The cell culture supernatant was collected. The pro-inflammatory cytokines IL-2 and IL-12p70 secreted by DCs were measured using an ELISA kit. Flow cytometry was used to examine the maturation of DC cells after staining with anti-CD80-APC, anti-CD86-PE, and anti-CD11c-FITC antibodies.

[0158] The effect of the system triggering tumor immunogenic cell death under ultrasound was detected by CLSM as Figure 6 shown. As can be seen from Figure 6 , under ultrasound stimulation, the MHS nanosystem could significantly promote tumor immunogenic cell death.

[0159] The in vitro immune activation effect was analyzed by flow cytometry and ELISA as Figure 7 shown. As can be seen from Figure 7 , under ultrasound stimulation, the MHS nanosystem could significantly promote immune activation.

[0160] 6. Verification of the immune activation ability of LGG

[0161] Six 4T1 tumor-bearing mouse models were established and randomly divided into the LGG group and the control group. When the tumor volume reached 200 mm3, RNA sequencing of the tumors was performed (as Figure 8 shown) to explore the potential biological mechanisms by which LGG promotes the therapeutic effect. As can be seen from Figure 8As can be seen, transcriptome sequencing shows that LGG can activate multiple immune-related signaling pathways.

[0162] 7. Evaluation of the combined treatment effect of the cocktail therapy:

[0163] 4T1 tumor cells (1×106) were injected into the axilla of female Balb / c mice (~20 g) to establish a xenograft tumor model. These mice were randomly divided into 8 groups (n = 5): control (200 μL, PBS), LGG (200 μL, LGG = 1×107 CFU), MHS (200 μL, MHS = 10 mg / kg), LGG-MHS (200 μL, LGG = 1×107 CFU, MHS = 10 mg / kg), MHS+US (200 μL, MHS = 10 mg / kg, US = 1.0 MHz, 1.0 W / cm2, 50% duty cycle, 5 minutes), LGG-MH+US (200 μL, LGG = 1×107 CFU, MH = 10 mg / kg, US = 1.0 MHz, 1.0 W / cm2, 50% duty cycle, 5 minutes), LGG-MHI+US (200 μL, LGG = 1×107 CFU, MHI = 10 mg / kg, US = 1.0 MHz, 1.0 W / cm2, 50% duty cycle, 5 min), LGG-MHS+US (200 μL, LGG = 1×107 CFU, MHS = 10 mg / kg, US = 1.0 MHz, 1.0 W / cm2, 50% duty cycle, 5 minutes). The above drugs were injected on days 7, 9, 11, and 13 respectively, and the ultrasound treatment groups were irradiated with ultrasound on days 8, 10, 12, and 14 respectively. During days 7 - 21, the tumor volume and body weight of the mice were measured every 2 days. The tumor volume was calculated according to the formula (tumor length) × (tumor width)2 / 2. Immune memory effect: To study memory T cells, spleen tissues from mice in different groups were collected and stained with anti-CD8-APC, anti-CD3-FITC, anti-CD44-PE, and anti-62L APC-Cy7 antibodies. Flow cytometry was used to isolate and analyze effector memory T cells (CD3+CD8+CD44+CD62L-, Tem and CD3+CD8+CD44+CD62L+, Tcm).

[0164] The tumor growth curves of different treatment groups are as Figure 9 shown, and as can be seen from Figure 9 it, ultrasound stimulation can significantly enhance the anti-tumor effect of LGG-MHS.

[0165] Flow cytometric analysis of effector memory T cells is as Figure 10 shown, and as can be seen from Figure 10It can be seen that the LGG-MHS system can increase the proportion of memory T cells and prevent tumor recurrence.

[0166] The above is the implementation scheme of the present invention designed for a case. It should be noted that for those of ordinary skill in the art, several improvements can be made without departing from the principle of the present invention, and these improvements should also be regarded as the protection scope of the present invention.

Claims

1. A probiotic CRISPR / Cas9 nanosystem, characterized in that, The described probiotic CRISPR / Cas9 nanosystem includes lactic acid bacteria and a CRISPR / Cas9 nanosystem; The lactic acid bacteria are loaded with the CRISPR / Cas9 nanosystem; The lactic acid bacteria are Lactobacillus rhamnosus; The CRISPR / Cas9 nanosystem includes a carrier, a photosensitizer, and a CRISPR / Cas9 system; The carrier is loaded with the photosensitizer and the CRISPR / Cas9 system; The photosensitizer is hematoporphyrin monomethyl ether.

2. The probiotic CRISPR / Cas9 nanosystem according to claim 1, wherein The CRISPR / Cas9 nanosystem is prepared by the following method, and the method includes: Mix and stir the photosensitizer, 2-methylimidazole, and zinc nitrate, centrifuge, and wash to obtain MH; Incubate MH with the CRISPR / Cas9 system to obtain the CRISPR / Cas9 nanosystem.

3. A method for preparing the probiotic CRISPR / Cas9 nanosystem as described in claim 1, characterized in that, The method includes: Mix and stir the photosensitizer, 2-methylimidazole, and zinc nitrate, centrifuge, and wash to obtain MH; Incubate MH with the CRISPR / Cas9 system to obtain the CRISPR / Cas9 nanosystem; Mix and stir the CRISPR / Cas9 nanosystem with lactic acid bacteria to obtain the probiotic CRISPR / Cas9 nanosystem.

4. A composition, characterized in that, The described composition includes the probiotic CRISPR / Cas9 nanosystem as claimed in claim 1.

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