Application of host-sourced aFRK mRNA in preparation of medicine for resisting bacteremia caused by drug-resistant staphylococcus aureus

Abstract

The invention discloses an application of host-derived aFRK mRNA in preparation of drugs for resisting bacteremia caused by drug-resistant staphylococcus aureus. MRNA of mouse-derived FRK activated mutant protein aFRK is synthesized in vitro and is introduced into a host cell through a lipid nanoparticle (LNP) delivery system, and the host-derived aFRK mRNA can be used for preparing drugs for resisting bacteremia caused by drug-resistant staphylococcus aureus. The macrophage can obviously enhance the killing ability of the macrophage on intracellular drug-resistant staphylococcus aureus, listeria monocytogenes and salmonella, so that infectious bacteria are effectively removed, bacteremia symptoms are improved, the survival rate is increased, and the macrophage has important clinical application value.

Description

TECHNICAL FIELD

[0001] The present application relates to the cross field of gene therapy and anti-infection therapy in the field of biotechnology, in particular to an activated mutant protein aFRK of mouse-derived FRK, an mRNA encoding aFRK, a lipid nanoparticle encapsulated preparation containing the aFRK mRNA, and further relates to the use of the lipid nanoparticle encapsulated preparation of aFRK mRNA in the preparation of a medicament for resisting sepsis caused by drug-resistant Staphylococcus aureus. BACKGROUND

[0002] The 2019 Global Burden of Disease report shows that among the 85 major human pathogens, Staphylococcus aureus (SA) ranks fifth, being the third largest pathogen after Mycobacterium tuberculosis and Streptococcus pneumoniae. Influenced by the continuous aggravation of antibiotic resistance, the number of deaths caused by SA infection has increased by 90% since 1990, making it the leading cause of death from bacterial infection, with more than 1 million deaths worldwide each year. Among them, SA sepsis (SAB) is a major fatal clinical phenotype, with a mortality rate of more than one-fourth within three months. At present, the clinical management of SAB is highly dependent on antibiotics, but the laboratory antibiotic resistance evolution model shows that SA is prone to develop resistance to such drugs, indicating that future clinical treatment will face great challenges.

[0003] Host-directed therapy (HDT) targeting host immunity brings new hope for the treatment of drug-resistant bacterial infections, as it is less likely to induce pathogen resistance. However, due to the lack of proven key targets that can enhance host anti-infection immunity, the development of SA-related HDT is still severely limited. Therefore, identifying new host molecules that can regulate host anti-SA immunity is expected to break the dependence on traditional antibiotics and provide a new treatment strategy for drug-resistant SA infection.

[0004] Liver-resident macrophages (i.e. Kupffer cells, KCs) are the main effector cells for capturing and clearing SA in the bloodstream. Studies have shown that in a mouse SAB model, KCs can phagocytose more than 90% of SA within 30 minutes, highlighting their strong immune defense effect. However, studies including those by our team have confirmed that SA can regulate the inflammatory response, autophagy, and phagolysosome maturation of macrophages through the secretion of effector factors, enabling intracellular survival and replication, and thus breaking through the immune barrier mediated by macrophages. Therefore, the interaction between KCs and SA directly determines the progression and prognosis of sepsis. In this context, identifying key host molecules that can enhance the bactericidal activity of macrophages against intracellular SA is a core strategy to help the host win the battle in immune defense against SA, and is also an important direction for the development of new antibacterial drugs.

[0005] Fyn-related kinase (FRK) is a non-receptor tyrosine kinase. Previous studies mainly focused on its regulatory role in breast cancer, colon cancer, liver cancer and other malignant tumors, while the functional research in the field of infection immunity is relatively scarce, especially the role in macrophage anti-SA infection has not been reported. At the same time, although mRNA technology has been widely used in vaccine development, the strategy of using host-derived functional mRNA combined with delivery system to treat bacteremia has not been reported. Based on this, the present application first proposes that by encapsulating aFRK mRNA with LNP, it is targeted to deliver to macrophages to enhance their killing ability to intracellular drug-resistant SA, providing a new host-oriented strategy for the treatment of drug-resistant SA bacteremia. SUMMARY

[0006] Therefore, the present application aims to provide a mouse-derived FRK activating mutant protein aFRK, the second aim of the present application is to provide a mRNA encoding the activating mutant protein aFRK, the third aim of the present application is to provide a lipid nanoparticle encapsulated preparation comprising the aFRK mRNA, the fourth aim of the present application is to provide a method for treating bacterial infection, the fifth aim of the present application is to provide the use of the lipid nanoparticle encapsulated preparation of the aFRK mRNA in the preparation of a drug for enhancing the clearance ability of macrophages to intracellular bacteria, the sixth aim of the present application is to provide the use of the lipid nanoparticle encapsulated preparation of the aFRK mRNA in the preparation of a drug for resisting bacteremia caused by drug-resistant Staphylococcus aureus, and the seventh aim of the present application is to provide the use of the lipid nanoparticle encapsulated preparation of the aFRK mRNA in the preparation of a drug for resisting infection of Listeria monocytogenes or Salmonella typhimurium.

[0007] To achieve the above-mentioned purposes, the present application provides the following technical solutions: 1. A mouse-derived FRK activating mutant protein aFRK, the amino acid sequence of the activating mutant protein aFRK is shown as SEQ ID NO. 3, which is obtained by mutating the amino acid at position 504 of the wild-type mouse FRK shown as SEQ ID NO. 1 from tyrosine to phenylalanine.

[0008] 2. A mRNA encoding the activating mutant protein aFRK, the preparation method comprising the following steps: constructing the nucleotide sequence encoding the activating mutant protein aFRK shown as SEQ ID NO. 5 to a prokaryotic expression vector, then amplifying the recombinant plasmid by E. coli, and extracting and purifying the recombinant plasmid; using the linearized recombinant plasmid as a template, in vitro transcription is carried out by T7 RNA polymerase, and after adding a 5' end cap structure and a 3' end poly(A) tail, DNAase is used to remove residual DNA template, and high-purity aFRK mRNA is obtained after purification.

[0009] 3. The lipid nanoparticle-encapsulated formulation comprising the aFRK mRNA, wherein the lipid nanoparticle comprises an ionizable cationic lipid, a neutral helper phospholipid, a PEG-modified phospholipid, and cholesterol.

[0010] 4. A method of treating a bacterial infection, comprising administering to an individual in need thereof an effective amount of the lipid nanoparticle-encapsulated formulation.

[0011] In some embodiments of the present application, the bacterial infection is an infection caused by drug-resistant Staphylococcus aureus, Listeria monocytogenes, or Salmonella.

[0012] In some embodiments of the present application, the route of administration is intravenous injection.

[0013] 5. Use of the lipid nanoparticle-encapsulated formulation of the aFRK mRNA in the preparation of a medicament for enhancing the ability of macrophages to clear intracellular bacteria, wherein the intracellular bacteria are one or more of drug-resistant Staphylococcus aureus, Listeria monocytogenes, and Salmonella.

[0014] 6. Use of the lipid nanoparticle-encapsulated formulation of the aFRK mRNA in the preparation of a medicament for resisting bacteremia caused by drug-resistant Staphylococcus aureus.

[0015] 7. Use of the lipid nanoparticle-encapsulated formulation of the aFRK mRNA in the preparation of a medicament for resisting infection by Listeria monocytogenes or Salmonella.

[0016] The present application has the beneficial effects that the present application first uses a host-derived aFRK mRNA for the treatment of drug-resistant Staphylococcus aureus infection, and constructs a brand-new host-oriented treatment strategy, thereby avoiding the problem of drug resistance induced by traditional antibiotics. The present application realizes efficient translation and expression in macrophages and mice in vivo through a lipid nanoparticle delivery system, and quickly exerts bactericidal activity. The present application is not only effective against drug-resistant Staphylococcus aureus, but also can clear other intracellular bacteria such as Listeria monocytogenes (hereinafter referred to as LM) and Salmonella Typhimurium (hereinafter referred to as ST), and thus has a wide range of applications. BRIEF DESCRIPTION OF DRAWINGS

[0017] In order to make the purpose, technical solutions and beneficial effects of the present application clearer, the present application provides the following drawings for illustration: Figure 1 The bactericidal function screening results of the overexpression of SFK family-related molecules in RAW264.7 cells confirm that FRK is a key molecule for killing SA in cells; Figure 2Comparison of phagocytosis of SA by wild type and FRK knockout macrophages Figure 3 Detection of clearance efficiency of intracellular SA by wild type and FRK knockout macrophages Figure 4 Comparison of liver bacterial load after infection of wild type and FRK liver conditional knockout mice with SA Figure 5 Verification of kinase activity of activated mutant aFRK and inactivated mutant iFRK Figure 6 Results of encapsulation rate of LNP-encapsulated aFRK and iFRK mRNA Figure 7 Results of particle size determination of LNP-encapsulated aFRK and iFRK mRNA Figure 8 Safety evaluation results of LNP-delivered aFRK and iFRK mRNA in macrophages (A is the CCK8 detection of cell viability results) and mice (B is the H-E staining results of each tissue of mice) Figure 9 Functional verification of LNP-delivered aFRK and iFRK mRNA in wild type macrophages after infection of SA, LM and ST (A is the experimental flowchart; B is the Western blot detection of aFRK and iFRK mRNA expression; C is the intracellular bacteria clearance effect) Figure 10 Immunohistochemical detection results of aFRK and iFRK mRNA expression in liver macrophages of wild type mice after tail vein injection of LNP-encapsulated aFRK and iFRK mRNA Figure 11 Influence of LNP-delivered aFRK and iFRK mRNA on liver bacterial load in wild type mice after infection of SA, LM and ST Figure 12 Evaluation of survival rate protection effect of LNP-delivered aFRK and iFRK mRNA on SA, LM and ST infected mice DETAILED DESCRIPTION

[0018] The present application will be further described below in conjunction with the drawings and specific examples, so that those skilled in the art can better understand the present application and implement it, but the examples are not intended to limit the present application.

[0019] Example 1 The host FRK molecule has the effect of resisting Staphylococcus aureus in vivo and in vitro. The FRK is obtained: through experiments, it is found that 11 SFK family members are overexpressed in macrophage RAW264.7, and it is found through intracellular bactericidal screening that the FRK has the most significant effect on the enhancement of macrophage clearance of intracellular SA Figure 1 , indicating that FRK is a key molecule in the SFK family that regulates resistance to SA infection. Phagocytic function verification shows that after FRK knockout (FRK - / - ), the phagocytosis of macrophages on SA has no effect Figure 2 , indicating that FRK does not participate in regulating the phagocytosis of macrophages on SA. The intracellular bactericidal results show that compared with wild-type macrophages, the killing ability of FRK - / - macrophages on intracellular SA is significantly weakened Figure 3 , confirming that FRK is a key regulatory molecule for macrophages to clear intracellular SA. Animal in vivo studies show that compared with FRK conditional knockout control mice (FRK [flox / flox] , FRK-Flox), the in vivo killing ability of liver-specific FRK conditional knockout mice (FRK [flox / flox, IRES-iCre] , FRK-CKO) on SA is significantly weakened Figure 4 , further proving that FRK plays an important role in host resistance to SA infection. The above in vivo and in vitro results show that FRK is a key molecule for the host to resist Staphylococcus aureus infection. The amino acid sequence of the FRK is shown as SEQ ID NO. 1, and the nucleotide sequence of the FRK is shown as SEQ ID NO. 2.

[0020] FRK amino acid sequence: MGSVCVRLWAYLQPFLPCWSQEADKSVVIENPGAFCPPEAPRSQEPERSHGQYFVALFDYQARTAEDLSFRAGDKLQVLDTSHEGWWLARHLEKKGTGLGQQLQGYIPSNYVAEDRSLQAEPWFFGAIKRADAEKQLLYSENQTGAFLIRESESQKGDFSLSVLDEGVVKHYRIRRLDEGGFFLTRRKVFSTLNEFVNYYTTTSDGLCVKLEKPCLKIQVPTPFDLSYKTADQWEIDRNSIQLLKRLGSGQFGEVWEGLWNNTTPVAVKTLKPGSMDPNDFLREAQIMKSLRHPKLIQLYAVCTLEDPIYIITELMRHGSLQEYLQNDGGSKIHLIQQVDMAAQVASGMAYLESQNYIHRDLAARNVLVGEHNIYKVADFGLARVFKVDNEDIYESKHEIKLPVKWTAPEAIRTNKFSIKSDVWSFGILLYEIITYGKMPYSGMTGAQVIQMLSQNYRLPQPSNCPQQFYSIMLECWNVEPKQRPTFETLHWKLEDYFETDCSYSDTNNFINDYKDDDDK (SEQ ID NO. 1) FRK protein-encoding DNA sequence: Example 2 Construction of mutant vectors of aFRK and iFRK and verification of enzyme activity: Construction of mutant vectors of aFRK and iFRK: Using site-directed mutagenesis technology, the Y504 of FRK tyrosine kinase (SEQ ID NO. 1~2) was mutated to phenylalanine (F) to obtain the activation mutant (aFRK, SEQ ID NO. 3), and the K269 of FRK was mutated to arginine (R) to obtain the inactivation mutant (iFRK, SEQ ID NO. 4).

[0021] Plasmid construction process: After artificially synthesizing the DNA sequences of the above-mentioned site-directed mutation aFRK and iFRK (SEQ ID NO. 5~6), the pcDNA3.1 vector and the artificially synthesized target gene fragment were double-digested by restriction endonuclease EcoRI and XhoI, and the linearized vector fragment and the target gene fragment were recovered. The two were directionally connected by DNA ligase to construct a recombinant expression vector. The ligation product was transformed into E. coli, and positive clones were screened with ampicillin resistance. The E. coli containing the recombinant plasmid was amplified, and the plasmid was purified by extraction to obtain the iFRK and aFRK mutant expression plasmids.

[0022] Amino acid sequence of aFRK: MGSVCVRLWAYLQPFLPCWSQEADKSVVIENPGAFCPPEAPRSQEPERSHGQYFVALFDYQARTAEDLSFRAGDKLQVLDTSHEGWWLARHLEKKGTGLGQQLQGYIPSNYVAEDRSLQAEPWFFGAIKRADAEKQLLYSENQTGAFLIRESESQKGDFSLSVLDEGVVKHYRIRRLDEGGFFLTRRKVFSTLNEFVNYYTTTSDGLCVKLEKPCLKIQVPTPFDLSYKTADQWEIDRNSIQLLKRLGSGQFGEVWEGLWNNTTPVAVKTLKPGSMDPNDFLREAQIMKSLRHPKLIQLYAVCTLEDPIYIITELMRHGSLQEYLQNDGGSKIHLIQQVDMAAQVASGMAYLESQNYIHRDLAARNVLVGEHNIYKVADFGLARVFKVDNEDIYESKHEIKLPVKWTAPEAIRTNKFSIKSDVWSFGILLYEIITYGKMPYSGMTGAQVIQMLSQNYRLPQPSNCPQQFYSIMLECWNVEPKQRPTFETLHWKLEDYFETDCSFSDTNNFINDYKDDDDK (SEQ ID NO. 3).

[0023] iFRK amino acid sequence: MGSVCVRLWAYLQPFLPCWSQEADKSVVIENPGAFCPPEAPRSQEPERSHGQYFVALFDYQARTAEDLSFRAGDKLQVLDTSHEGWWLARHLEKKGTGLGQQLQGYIPSNYVAEDRSLQAEPWFFGAIKRADAEKQLLYSENQTGAFLIRESESQKGDFSLSVLDEGVVKHYRIRRLDEGGFFLTRRKVFSTLNEFVNYYTTTSDGLCVKLEKPCLKIQVPTPFDLSYKTADQWEIDRNSIQLLKRLGSGQFGEVWEGLWNNTTPVAVRTLKPGSMDPNDFLREAQIMKSLRHPKLIQLYAVCTLEDPIYIITELMRHGSLQEYLQNDGGSKIHLIQQVDMAAQVASGMAYLESQNYIHRDLAARNVLVGEHNIYKVADFGLARVFKVDNEDIYESKHEIKLPVKWTAPEAIRTNKFSIKSDVWSFGILLYEIITYGKMPYSGMTGAQVIQMLSQNYRLPQPSNCPQQFYSIMLECWNVEPKQRPTFETLHWKLEDYFETDCSYSDTNNFINDYKDDDDK (SEQ ID NO. 4).

[0024] aFRK protein-encoding DNA sequence: iFRK protein-encoding DNA sequence: Sequence verification: The constructed aFRK and iFRK expression plasmids were sequenced and verified to confirm that all mutation sites were correct.

[0025] Protein expression and enzyme activity verification: (1) Resuscitate HEK293T cells: Take the frozen cells out of liquid nitrogen, melt in a 37°C water bath, then transfer into a 50 mL centrifuge tube, add 10 mL high-sugar DMEM complete medium, mix well, centrifuge at 800 rpm for 5 min, discard the supernatant. Resuspend with 2 mL medium, then add to 10 mL, transfer to a 100 mm culture dish, incubate at 5% CO2, 37°C.

[0026] (2) Cell plating: When the cells grow to 90%, discard the supernatant, add 1 mL 0.25% trypsin and digest for 30 s, then discard, add 5 mL medium to blow the cells down, transfer to a centrifuge tube and centrifuge at 800 rpm for 5 min; resuspend the medium after discarding the supernatant and count, plate at a density of 5x10 6 / dish in a 60 mm culture dish, and culture for 2 days before transfection.

[0027] (3) Plasmid transfection: Take 3 1.5 mL EP tubes, add 250 μL blank DMEM to each, and add 4 μg plasmid to each. Prepare PEI diluent: PEI stock volume = total amount of plasmid in each tube x 3 x (n+1) (n is the number of samples), required blank DMEM = 250 μL x (n+1) - PEI stock volume, mix well and add to the EP tube with plasmid, 250 μL per tube, mix well and incubate at room temperature for 30 min. Slowly add the plasmid suspension along the wall to the pre-plated cells, mix gently. Replace the complete medium after 4-6 h.

[0028] (4) Collect cells and run gel: 48 h after transfection, discard the supernatant, wash with pre-cooled PBS 3 times, add 500 μL lysis buffer to each dish, and lyse at 4°C on a shaker for 30 min. Transfer the lysis buffer to an EP tube and centrifuge at 13000 rpm for 10 min at 4°C. Transfer the supernatant to a new EP tube, add 45 μL and 15 μL 4x loading buffer, boil at 100°C for 10 min, and store at 4°C for electrophoresis detection. Add 10 μL Myc-Beads to the remaining supernatant and rotate overnight at 4°C. The next day, use a magnetic stand to adsorb the magnetic beads, discard the supernatant, wash with 1 mL 0.1% PBST 3 times for 10 min each time, and finally add 60 μL 1x loading protein buffer and boil at 100°C for 10 min. Western blot to detect FRK mutant protein expression level and phosphorylation state, method steps as follows: ① Preparation of SDS-PAGE gel: 50 mL centrifuge tube, add separation gel A and B liquid at 1:1, add appropriate amount of 10% APS, mix well and pour into the electrophoresis mold to 1.5 cm from the top. At the same time, mix the concentrated gel A and B liquid at 1:1, add appropriate amount of 10% APS, and pour directly on the upper layer of the separation gel. Insert the comb, stand for 15 min until completely solidified, fix the gel on the electrophoresis gel plate, and place in the electrophoresis tank containing the electrophoresis buffer, and pull out the comb.

[0029] ② Electrophoresis: constant voltage 80V for concentrated gel, constant voltage 200V for separation gel, bromophenol blue to the edge of the gel bottom; ③ Membrane transfer: constant current 400mA, ice bath membrane transfer for 30min; ④ 5% skim milk powder room temperature blocking PVDF membrane 1h, 1% TBST rinsing 3 times, adding corresponding antibody (rabbit anti-mouse protein tyrosine phosphorylation pan antibody, rabbit anti-mouse anti-Myc tag antibody) 4℃ incubation overnight; ⑤ After recovering the antibody, rinse with TBST for 3 times, add secondary antibody (goat anti-rabbit HRP labeled antibody) and incubate at room temperature for 1h; ⑥ TBST rinsing 3 times, ECL luminescent solution development, E-Blot imaging system photographing.

[0030] The results are shown in Figure 5 aFRK protein has a high level of autophosphorylation, while iFRK protein has a significantly reduced autophosphorylation level. It is confirmed that aFRK is an activated mutant, and iFRK is an inactivated mutant.

[0031] Example 3 Preparation of LNP aFRK and iFRK mRNA The preparation method of the LNP mRNA is as follows: Template preparation: the aFRK and iFRK plasmids prepared in Example 2 with T7 promoter were single-enzymatically cut with restriction enzyme XhoI, and the system was as follows: 10 μL of 10×Quick Cut Buffer, 2 μg of aFRK or iFRK plasmid DNA, 2 μL of XhoI were contained in 100 μL, and the rest was supplemented with nuclease-free water. Incubate in a 37°C water bath for 2h. Prepare 1% agarose gel, and spot the original plasmid and the single-enzymatically cut plasmid for detection to ensure that the enzyme cutting is successful. Then purify the enzyme cutting product with a PCR purification kit to obtain a linearized plasmid.

[0032] In vitro mRNA transcription: (1) The transcription system is as follows: 10 μL of 2×ARCA / NTP Mix, 1 μg of linear plasmid DNA, 2 μL of T7 RNA Polymerase Mix are contained in 20 μL, and the rest is supplemented with nuclease-free water. Mix well, centrifuge at low speed, and incubate at 37°C for 4h.

[0033] (2) Add 2 μL DNase I, continue incubation at 37°C for 15 min to remove template DNA.

[0034] (3) Add 1 / 2 volume of pre-cooled LiCl, precipitate at -20°C overnight; (4) Centrifuge at 13000 g for 5 min, remove supernatant; (5) Wash 3 times with pre-cooled 75% ethanol, and finally evaporate ethanol for 5 min; (6) Freeze the RNA on dry ice, dissolve at room temperature, and vortex again, repeat 2-3 times until the RNA is completely hydrated.

[0035] mRNA capping: (1) First add 60 μg of RNA and nuclease-free water, incubate at 65°C for 5 min, and quickly place on ice; (2) Add EZ Cap TM reagent AG and N1-Methylpseudo-UTP, etc., total volume 100 μL, incubate at 37°C for 1 h; (3) Add 1 / 2 volume of pre-cooled LiCl, precipitate at -20°C overnight; (4) Centrifuge at 13000 g for 5 min, remove supernatant; (5) Wash 3 times with pre-cooled 75% ethanol, and finally evaporate ethanol for 5 min; (6) Freeze the RNA on dry ice, dissolve at room temperature, and vortex again, repeat 2-3 times until the RNA is completely hydrated.

[0036] mRNA Poly (A) tailing: (1) Add mRNA and Poly (A) tail reagent, etc., total volume 25 μL, incubate at 37°C for 1 h; (2) Add 1 / 2 volume of pre-cooled LiCl, precipitate at -20°C overnight; (3) Centrifuge at 13000 g for 5 min, remove supernatant; (4) Wash 3 times with pre-cooled 75% ethanol, and finally evaporate ethanol for 5 min; (5) Freeze the RNA on dry ice, dissolve at room temperature, and vortex again, repeat 2-3 times until the RNA is completely hydrated.

[0037] mRNA purification: Using monodisperse superparamagnetic beads coupled with oligo-dT sequence, based on the base pairing between oligo-dT sequence and mRNA Poly (A) tail, high-purity mRNA is separated by magnetic sorting.

[0038] mRNA coating: (1) mRNA is delivered by APExBIO's proprietary lipid nanoparticle (LNP) technology, which is usually composed of ionizable cationic lipids, neutral helper phospholipids, PEG-modified phospholipids, and cholesterol mixed in a specific ratio; (2) iFRK and aFRK mRNA are rapidly mixed and encapsulated with LNP based on precise control program and Feather microfluidic chip technology using NanoDispatcher™ L Nanomixer; (3) The particle size, PDI, and Zeta potential of LNP are determined by dynamic light scattering method; (4) The encapsulation efficiency of mRNA is determined by fluorescent dye method.

[0039] The results show that the mRNA encapsulation efficiency is ≥90% ( Figure 6 ), and the particle size is 80-120 nm ( Figure 7 ).

[0040] Example 4 Cell viability and safety evaluation of LNP mRNA aFRK and iFRK CCK8 assay cell viability: (1) RAW264.7 cells are seeded in a 96-well plate at 20000 cells per well and cultured in a cell incubator for 24 h; (2) Add 10 μL (containing 125 ng) of LNP-iFRK and LNP-aFRK mRNA to the wells and treat for 12 h; (3) Add 10 μL CCK-8 Plus to each well; (4) Incubate the plate in the incubator for 0.5-3 h (the specific time can be adjusted according to the specific experiment); (5) Then shake the plate well using a microplate reader and detect the absorbance at 450 nm; (6) Data analysis: cell survival rate (%) = [(As-Ab) / (Ac-Ab)] x 100% As = absorbance of experimental wells (absorbance of wells containing cells, culture medium, CCK-8 Plus, and LNP-mRNA) Ab = absorbance of blank wells (absorbance of wells containing culture medium and CCK-8 Plus) Ac = absorbance of control wells (absorbance of wells containing cells, culture medium, and CCK-8 Plus) The results are as follows Figure 8, A showed that LNP-iFRK and LNP-aFRK mRNA had no significant effect on the proliferation activity of RAW264.7 cells, and did not show cytotoxicity, indicating that the preparation had good safety at the cellular level.

[0041] H-E staining was used to observe the damage of each tissue: (1) SPF level male C57BL / 6 mice aged 6-8 weeks and weighing 18-22 g were injected with 10 μg of LNP-iFRK and LNP-aFRK mRNA via the tail vein. Seven days after injection, the mouse heart, liver, spleen, lung, and kidney tissues were taken and fixed with 4% paraformaldehyde for HE staining pathological sections; (2) After paraffin sections were deparaffinized with environmentally friendly deparaffinizing agent for 3 times, they were treated with anhydrous ethanol, 95% ethanol, 85% ethanol, and 75% ethanol for 5 min each, and then rinsed with tap water for 1 min; (3) The sections were stained with hematoxylin staining solution Harris for 4 min, and then rinsed with tap water for 2 min until no excess dye was eluted from the sections; (4) The sections were differentiated with 0.8% hydrochloric acid alcohol for 2 s, rinsed with tap water, or blued with lithium carbonate aqueous solution, and then rinsed with water for 2 min; (5) The sections were stained with eosin staining solution for 20 s without water washing, and then adjusted in color with 95% ethanol for 5 s and dehydrated with anhydrous ethanol for 2 min each time; (6) Finally, the sections were sealed with environmentally friendly transparent agent, and then scanned by Nanozoomer® S360 imaging system.

[0042] The results are shown in Figure 8 , B showed that after the mice were injected with LNP-iFRK and LNP-aFRK mRNA via the tail vein, the H-E staining results of the key organ tissues such as heart, liver, spleen, lung, and kidney showed that the staining results of each tissue did not suggest abnormal lesions, and were completely consistent with the detection results of SPF mice (standard control without specific pathogen), indicating that the preparation had good safety.

[0043] Example 5 Effects of aFRK and iFRK on in vitro clearance of SA, Listeria LM, and ST According to Figure 9 , A experimental flow chart, the effects of aFRK and iFRK on in vitro clearance of SA, LM, and ST were compared.

[0044] BMDM cell separation and induced differentiation: (1) After the C57BL / 6 wild mice were executed by decapitation, they were immediately soaked in 75% ethanol for 10 min for disinfection; under sterile operating conditions, the femur and calf bone of the hind leg were separated, and the both ends of the bone were removed with sterile scissors; (2) Bone marrow cavity was washed with 10 mL complete DMEM (containing L-glutamine and sodium pyruvate), and the lavage fluid was collected and centrifuged at 4°C and 800 rpm for 5 min; after the supernatant was discarded, an appropriate amount of red blood cell lysis solution was added to the precipitate, which was placed at room temperature for 5 min, and then complete DMEM (containing 10% Moregate fetal bovine serum, 1% fungal antibiotics) was added to terminate lysis, and the precipitate was centrifuged at 800 rpm for 10 min, and then washed with 20 mL DMEM for 2 times; (3) Resuspend the cells with complete DMEM containing 10% FBS, 1% antibiotics and 20% L929 cell culture supernatant (or 10 ng / mL M-CSF), count and inoculate 1×10 7 cells / dish in TC-treated flat-bottom cell culture plates to start differentiation induction (recorded as day 1 on the same day); (4) On the 4th day of differentiation, the original culture medium was discarded and replaced with 10 mL of fresh complete culture medium, and the culture was continued at 37°C in a 5% CO2 incubator for 2 days; on the 6th day, the cells were collected by blowing with complete culture medium, centrifuged at 800 rpm for 10 min, and the precipitate was resuspended with DMEM containing 10% FBS and 1% antibiotics, and plated at a density of 5×10 5 cells / well in 24 and 12 well plates for standby.

[0045] Detection of expression of BMDM cells transfected with LNP iFRK and aFRK mRNA: (1) After BMDMs were transfected with LNP iFRK and aFRK mRNA for 6 h, 100 μL of 1× loading protein buffer was added to lyse the cells, and the cells were collected and boiled at 100°C for 10 min.

[0046] (2) Western blot was used to detect the expression of FRK mutant proteins, and the specific operation of this step was consistent with that of Example 2.

[0047] The results are shown in Figure 9 , B, iFRK and aFRK are highly expressed in BMDMs.

[0048] Strain recovery: (1) Strain streak culture: SA (ATCC BAA-1556), LM (ATCC BAA-679) and ST (ATCC 39184) strains were taken out from the -80°C low-temperature refrigerator, respectively, and were streaked on TSB, BHI and LB solid plates using the three-line method for overnight culture to obtain single colonies; (2) Single colony expansion culture: the next day, single colonies were inoculated into TSB, BHI and LB liquid culture medium, respectively, and cultured at 37°C and 220 rpm on a shaking bed overnight. (3) OD600nm measurement: On the third day of culture, each strain was inoculated into the corresponding fresh medium at a ratio of 1:100, and continued to be cultured at 37°C, 220 rpm for 3h. After the end of culture, centrifugation was performed at 4°C, 6000 rpm for 5 min, and the supernatant was discarded. The bacterial cells were washed with sterile PBS for 3 times. The bacterial cells were resuspended with PBS, and the absorbance at OD600nm was detected. The concentration of the bacterial cells was calculated and adjusted for subsequent experiments.

[0049] Infection of BMDM cells and plate: (1) Cell treatment and infection: ① When BMDMs were infected with SA, the multiplicity of infection MOI = 5, and the infection time was 30 min. After infection, the cells were treated with 10 ng / mL lysostaphin for 10 min. Then the cells were washed with PBS buffer for 3 times. Then the cells were supplemented with 25 μg / mL gentamicin in high glucose DMEM medium, and LNP-encapsulated iFRK and aFRK mRNA were added for transfection and culture for 12 h. ② When LM was infected, the multiplicity of infection MOI = 5, and the infection time was 30 min. When ST was infected, the multiplicity of infection MOI = 20, and the infection time was 60 min. After the above two bacterial infections, 100 μg / mL of gentamicin was used to remove extracellular bacteria, and then LNP-encapsulated iFRK and aFRK mRNA were added for transfection and culture for 12 h.

[0050] (2) Intracellular bacterial counting and data analysis: After infection, 200 μL of PBS containing 0.1% Triton-X100 was used to lyse the cells. The lysate was gradiently diluted and inoculated into the corresponding solid culture medium, and incubated at 37°C overnight. The next day, CFU counting was performed. The data was statistically analyzed and plotted using Excel and Garphpad Prism 10.5.0 software.

[0051] The results are shown in Figure 9 , C, compared with transfection of iFRK mRNA, transfection of aFRK mRNA can significantly enhance the clearance of intracellular SA, LM and ST by macrophages.

[0052] Example 6 Immunohistochemical results of aFRK and iFRK expression in mouse liver (1) Sample preparation: C57BL / 6 wild-type SPF mice were injected with LNP-encapsulated iFRK and aFRK mRNA (dose of 10 μg per mouse) via the tail vein. The mice were sacrificed 3h after injection, and the liver tissue was fixed with 4% paraformaldehyde for immunohistochemical detection. (2) Paraffin section dewaxing to water: the paraffin section was placed in the environmental protection dewaxing agent for 10 min, and then immersed in anhydrous ethanol, 95% ethanol, 75% ethanol for 5 min, respectively; washed with distilled water for 3 times, 3 min each time, and then immersed and washed.

[0053] (3) Antigen repair: high temperature and high pressure repair method was used, EDTA (pH 9.0) antigen repair solution was added into the high pressure pot, and the volume of the repair solution was appropriate to immerse the section. Loosely cover the lid, place it on the 2100 watt electromagnetic oven, heat to boiling, put the dewaxed and hydrated tissue section into the boiling repair solution, tightly cover the pot lid, and then install the pressure limiting valve after the air nozzle rises, adjust the power of the electromagnetic oven to 1200 watts, and then stop the fire after 90 seconds of timing after the high pressure pot sprays; use cold water to flush the high pressure pot until the air pressure drops, open the pot lid, and then take out the section after the repair solution naturally cools to room temperature (25-30℃, 1h), and then immerse the section in TBST buffer for 3 times, 5 min each time.

[0054] (4) Tissue circle: draw a circle around the tissue with a group of pens, and then place the section in TBST buffer.

[0055] (5) Blocking: drop 10% donkey serum, and incubate at 37℃ for 30 min.

[0056] (6) Incubation of primary antibody (F4 / 80 antibody): discard the serum, add F4 / 80 antibody stock solution to TBST buffer, prepare 1:4000 primary antibody working solution with a concentration of, mix with a shaking mixer, drop 50-100 μL of primary antibody working solution on each section, and incubate at 4℃ overnight.

[0057] (7) Incubation of secondary antibody (HRP labeled secondary antibody): take out the section the next day, and then place it at room temperature for 15 min to warm up, wash it with TBST buffer for 3 times, and then immerse and wash it for 3 times, 3 min each time. Take goat anti-rabbit IgG H&L (HRP) secondary antibody stock solution and add it to TBST buffer, prepare 1:4000 secondary antibody working solution with a concentration of, mix with a shaking mixer, drop 50-100 μL (according to the size of the tissue) of secondary antibody working solution on each section, and incubate at 37℃ for 45 min. Wash it with TBST buffer for 3 times, and then immerse and wash it for 3 times, 3 min each time.

[0058] (8) TSA staining: remove the TBST buffer, take CY5 tyramide stock solution and add it to 0.003% H2O2, prepare 1:600 CY5 tyramide working solution, drop 50-100 μl of CY5 tyramide working solution on each section, and incubate at room temperature for 10 min. After incubation, immerse and wash it with TBST for 3 times.

[0059] (9) Antibody elution: add citric acid repair solution to the repair box, and the repair solution should cover the section. Put the repair box into the microwave oven and repair for 5 min at medium heat. Take out the section and immerse in the TBST buffer for 3 min.

[0060] (10) Secondary blocking: drop 10% donkey serum and incubate at 37℃ for 30 min.

[0061] (11) Incubate the first antibody (Flag antibody): shake off the serum, take the FLAG antibody stock solution, add to the TBST buffer, prepare the first antibody working solution with a concentration of 1:1000, mix with the shaking homogenizer, drop 50-100 μL of the first antibody working solution on each section, and incubate at 4℃ overnight.

[0062] (12) Incubate the second antibody (fluorescently labeled secondary antibody): take out the section on the next day, place it at room temperature for 15 min for rewarming, wash with the TBST buffer for 3 times, and immerse for 3 times, each for 3 min. Take the Donkey anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor™ 488 secondary antibody stock solution, add to the TBST buffer, prepare the secondary antibody working solution with a concentration of 1:600, mix with the shaking homogenizer, drop 50-100 μL (depending on the size of the tissue) of the secondary antibody working solution on each section, and incubate at 37℃ for 45 min. Wash with the TBST buffer for 3 times, and immerse for 3 times, each for 3 min.

[0063] (13) Nucleus staining: discard the TBST buffer, drop 50-100 μl (depending on the size of the tissue) of the DAPI working solution (prepared by mixing the DAPI stock solution at 1:500) on each section, avoid light for 5 min of staining, and then wash with the TBST buffer.

[0064] (14) Mounting and preservation: mount with the fluorescent mounting medium, and preserve at 4℃ in the dark.

[0065] (15) Microscopic analysis: observe under the microscope, collect images, and analyze.

[0066] The results are shown in Table 1. Figure 10 Both iFRK and aFRK can be expressed in mouse liver macrophages.

[0067] Example 7 Study on the clearance effect of aFRK and iFRK on SA, LM and ST in vivo (1) Strain recovery and OD600nm determination: refer to the operation steps of Example 5 of the present application.

[0068] (2) Animal infection and treatment: C57BL / 6 wild-type SPF mice were infected with SA (dose of 2x10 7 CFU / mouse), LM (dose of 2x10 5 CFU / mouse) or ST (dose of 2x10 5 CFU / mouse) by tail vein injection, and 1 h after infection, the mice were injected with LNP-encapsulated iFRK and aFRK mRNA via the tail vein.

[0069] (3) Liver bacterial load detection: 12 h after infection, the mice were sacrificed and liver tissues were collected, homogenized with PBS buffer, and gradient diluted, and the diluted solution was inoculated on the corresponding medium for culture, and colony counting was performed to determine the bacterial load in the liver tissues.

[0070] As shown in Figure 11 , compared with injection of iFRK mRNA, injection of aFRK mRNA can significantly enhance the in vivo clearance ability of mice to SA, LM and ST.

[0071] Example 8 Evaluation of the protective effect of aFRK and iFRK on infected mice (1) Strain recovery and OD600nm determination: The experimental operation steps refer to Example 5 of the present application.

[0072] (2) Animal infection and treatment: C57BL / 6 wild-type SPF mice were infected with SA (dose of 5x10 7 CFU / mouse), LM (dose of 5x10 7 CFU / mouse) or ST (dose of 5x10 7 CFU / mouse) by tail vein injection, and 1 h after infection, the mice were injected with LNP-encapsulated iFRK and aFRK mRNA via the tail vein.

[0073] (3) Survival observation: The above treated mice were continuously observed for 7 days, and the survival rate was calculated.

[0074] The experimental results are shown in Figure 12 , compared with the control mice injected with iFRK mRNA, the mortality of the experimental mice injected with aFRK mRNA was significantly reduced.

[0075] In summary, the application finds that FRK is a key regulatory molecule for macrophages to clear intracellular SA through screening, and obtains an activating mutant aFRK (Y504F) and an inactivating mutant iFRK (K269R) of FRK by using site-directed mutagenesis technology; the activating mutant aFRK mRNA is encapsulated by LNP and targeted to be delivered to macrophages, and the aFRK mRNA can significantly enhance the clearance ability of bone marrow-derived macrophages to intracellular Staphylococcus aureus, Listeria monocytogenes and Salmonella; the iFRK and aFRK mRNA encapsulated by LNP are injected into mice infected with Staphylococcus aureus, Listeria monocytogenes and Salmonella through the tail vein, and it is found that, compared with the injection of iFRK mRNA, the injection of aFRK mRNA can significantly enhance the in vivo clearance ability of mouse liver tissue to Staphylococcus aureus, Listeria monocytogenes and Salmonella, and the injection of aFRK mRNA significantly reduces the mortality of experimental mice.

[0076] The above-described embodiments are only preferred embodiments of the present application, and the protection scope of the present application is not limited thereto. Any equivalent replacement or transformation made by those skilled in the art on the basis of the present application is within the protection scope of the present application. The protection scope of the present application is subject to the claims.

Claims

1. A mouse-derived FRK-activating mutant protein aFRK, characterized in that, The amino acid sequence of the activated mutant protein aFRK is shown as SEQ ID NO. 3, which is obtained by mutating the amino acid at position 504 of the wild-type mouse FRK shown as SEQ ID NO. 1 from tyrosine to phenylalanine.

2. An mRNA encoding the activated mutant protein aFRK according to claim 1, characterized in that, The preparation method comprises the following steps: constructing the nucleotide sequence encoding the activated mutant protein aFRK shown as SEQ ID NO. 5 to a prokaryotic expression vector, then amplifying the recombinant plasmid by E. coli, and extracting and purifying the recombinant plasmid; using the linearized recombinant plasmid as a template, in vitro transcription is performed by T7 RNA polymerase, and after adding a 5' end cap structure and a 3' end poly(A) tail, the residual DNA template is removed by DNase digestion, and high-purity aFRK mRNA is obtained after purification.

3. Lipid nanoparticle-encapsulated formulation comprising the aFRK mRNA of claim 2, characterized in that, The lipid nanoparticle comprises an ionizable cationic lipid, a neutral helper phospholipid, a PEG-modified phospholipid, and cholesterol.

4. A method of treating a bacterial infection, comprising administering to a subject in need thereof a therapeutically effective amount of a compound of claim 1. The application further provides a use of the lipid nanoparticle encapsulated preparation of claim 3 in the preparation of a medicament for preventing and treating a bacterial infection.

5. The method of claim 4, wherein, The bacterial infection is an infection caused by drug-resistant Staphylococcus aureus, Listeria monocytogenes, or Salmonella.

6. The method according to claim 4 or 5, characterized in that, The route of administration is intravenous injection.

7. Use of the lipid nanoparticle-encapsulated formulation of aFRK mRNA according to claim 3 for the manufacture of a medicament for enhancing the ability of macrophages to clear intracellular bacteria, characterized in that, The intracellular bacteria are one or more of drug-resistant Staphylococcus aureus, Listeria monocytogenes, and Salmonella.

8. Use of the lipid nanoparticle encapsulated preparation of aFRK mRNA of claim 3 in the preparation of a medicament for resisting bacteremia caused by drug-resistant Staphylococcus aureus.

9. Use of the lipid nanoparticle encapsulated preparation of aFRK mRNA of claim 3 in the preparation of a medicament for resisting an infection of Listeria monocytogenes or Salmonella.

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