Application of HGFmRNA LNP in the preparation of a drug for treating emphysema

Abstract

The present invention relates to an application of HGF mRNA LNP in preparing a drug for treating emphysema, belonging to the field of biomedicine. The present invention uses a lipid nanoparticle carrier (lipid nanoparticle, LNP) to deliver HGF factor to the lungs for the first time. Experiments have confirmed that HGF mRNA LNP has high expression efficiency in vivo and strong targeting to lung tissue, and can effectively promote the repair of lung tissue structure in mice with emphysema, and has the characteristics of minimal invasion and high efficiency.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular, to application of HGF mRNA LNP in preparing a medicine for treating emphysema. Background Art

[0002] Chronic obstructive pulmonary disease (COPD), abbreviated as COPD, is a chronic heterogeneous respiratory disease characterized by persistent respiratory symptoms and progressive airflow limitation. Due to irreversible airflow obstruction and progressive lung function decline, COPD is characterized by high mortality and disability, bringing huge economic burden to society. Current treatments for COPD, such as bronchodilators or inhaled glucocorticoids, may help alleviate the progression of COPD, but cannot reverse lung tissue damage or improve patients' quality of life. Among them, patients with emphysematous phenotypes present chronic pathophysiological changes, accompanied by decreased elasticity of distal small airways, destruction of lung tissue and alveolar walls, and ultimately causing excessive expansion of distal airways and permanent increase in lung volume. Lung tissue damage and enhanced catabolism are the main pathophysiological characteristics of the emphysematous phenotype of COPD. As the elastic tissue of the lung is destroyed, the elastic recovery force of the lung decreases, resulting in insufficient pressure to exhale gas during exhalation, ultimately causing gas trapping and excessive lung expansion.

[0003] Hepatocyte growth factor (HGF) has been shown to be essential for the development and maintenance of alveoli during embryonic and postnatal development in rodents. HGF was originally isolated from mature hepatocytes in primary culture and is a potent mitogen. It is mainly secreted by fibroblasts, mesenchymal cells, and smooth muscle cells and is involved in the repair process of various organs and tissues, such as liver, kidney, heart, and lung injuries. However, there are no reports on the use of HGF to treat emphysema, which may be related to its inadequate delivery technology. For example, invasive procedures such as surgery or osmotic pump injection increase the risk of infection and death. Injection of ASCs or HGF cDNA through the blood has the problem of low efficiency of HGF overexpression, while direct administration of HGF recombinant protein into the airway increases the degradation rate of HGF. Therefore, the development of a new drug carrier delivery system for HGF is of great significance to promote the regeneration of lung tissue structure in emphysema. Summary of the invention

[0004] In order to overcome the defects in the prior art, the present invention uses lipid nanoparticle carriers (LNP) for the first time to deliver HGF factor to the lungs. Experiments have confirmed that HGF mRNA LNP has high expression efficiency in vivo and strong targeting to lung tissue, and can effectively promote the repair of lung tissue structure in mice with emphysema, with the characteristics of minimally invasive and high efficiency.

[0005] Specifically, the present invention provides the following technical solutions:

[0006] The first aspect of the present invention provides a use of HGF mRNA LNP in the preparation of a drug for treating emphysema.

[0007] In one embodiment, the HGF mRNA LNP comprises the HGF ORF sequence as described in SEQ ID NO.1.

[0008] In one embodiment, the HGF mRNA LNP is prepared by the following method:

[0009] (3) preparing a lipid mixture: the lipid mixture includes SM102, Cholesterol, DMG-PEG, and DSPC;

[0010] (4) HGF mRNA was diluted with Citrate buffer and then added to the lipid mixture of lipidmix and EtOH.

[0011] Use a pipette to quickly pipette and mix to obtain HGF mRNA LNP.

[0012] In a preferred embodiment, SM102:Cholesterol:DMG-PEG:DSPC=5.88:18.56:4.68:9.84; lipidmix:EtOH:HGF mRNA:Citrate buffer (10 mM, pH 4)=20.16:1.44:9.6:55.2.

[0013] In one embodiment, the treatment comprises inhibiting emphysematous lung inflammation, promoting alveolar epithelial proliferation and inhibiting apoptosis.

[0014] Compared with the prior art, the present invention has the following significant improvements:

[0015] (1) In vitro transcription of mRNA is simpler than protein, has high operability, low cost, and is easy to industrialize, while protein synthesis is more expensive;

[0016] (2) Compared with DNA, which must enter the cell nucleus to initiate transcription, mRNA can be directly translated into protein in the cytoplasm, which takes effect faster. Its translation efficiency is much higher than that of DNA, and in theory it can be translated into any protein, which has the potential to treat a variety of diseases;

[0017] (3) When DNA and viruses overexpress target genes, there is a risk of inserting into the original genome of the cell and changing the genetic information, while mRNA only acts transiently when translating proteins in the cytoplasm and then rapidly degrades, without the risk of genome integration, and is safer;

[0018] (4) The experiment confirmed that the HGF mRNA LNP system has a high load encapsulation rate, high in vivo expression efficiency, and strong targeting to lung tissue, which provides key technical support for in vivo studies on HGF mRNA LNP promoting lung injury repair in emphysema. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] The accompanying drawings are used to provide a further understanding of the present invention and constitute a part of the specification. Together with the embodiments of the present invention, they are used to explain the present invention and do not constitute a limitation of the present invention. In the accompanying drawings:

[0020] Figure 1 SM102 is the best LNP for delivering mRNA to the lungs. (A) MC3, SM102, and ALC0315 LNPs were used to encapsulate luciferase mRNA and then delivered to the lung tissue by airway instillation. After 12 hours, the fluorescence signal was detected by bioluminescence imaging; (B) Bioluminescence imaging of simple mRNA delivery and luciferase mRNA encapsulated by different types of LNPs and then delivered to the lungs; (C) Statistical graph of fluorescence signal values.

[0021] Figure 2 SM102 LNP significantly improves the stability of mRNA in the lung. (A) SM102 LNP was used to encapsulate luciferase mRNA and then deliver it to lung tissue via airway instillation; (B) After airway delivery of luciferase mRNA, bioluminescence imaging was performed at 6h, 12h, 24h, and 48h; (C) Statistical graph of fluorescence signal values.

[0022] Figure 3 :The optimal dose test of SM102 LNP loaded with mRNA. (A) SM102 LNP was used to encapsulate 5μg, 15μg, and 30μg luciferase mRNA and then delivered to lung tissue by airway instillation; (B) After airway delivery of different doses of luciferase mRNA, bioluminescence imaging was performed at the 12h time point; (C) Statistical graph of fluorescence signal values.

[0023] Figure 4 :HGF mRNA-SM102 LNP can significantly improve the lung function of mice with emphysema. (A) Dynamic elasticity of the respiratory system; (B) Dynamic compliance of the respiratory system; (C) Quasi-static compliance; (D) Tissue elasticity; (E) Forced inspiratory volume; (F) Total lung capacity.

[0024] Figure 5 :HGF mRNA-SM102 LNP can significantly repair the abnormally enlarged alveolar cavity in emphysema mice. (A) Control group; (B) PPE-induced emphysema group; (C) Emphysema + Luc mRNA LNP treatment group; (D) Emphysema + HGF mRNA LNP treatment group; (E) Emphysema + HGF recombinant protein treatment group; (F) Semi-quantitative analysis of the average linear intercept of the alveoli. Figure 6 : Results of blood routine and blood biochemical analysis of mice. (A) Leukocytes; (B) Red blood cells; (C) Platelets; (D) Hemoglobin; (E) Lymphocytes; (F) Alkaline phosphatase; (G) Creatinine; (H) ALT; (I) AST; (J) Lactate dehydrogenase.

[0025] Figure 7 : Detection of inflammation in mouse lung tissue. RT-qPCR was used to detect the expression of inflammatory factors such as (A) IL6, (B) IL1β, and (C) TNFα in mouse lung tissue; ELISA was used to detect the expression of inflammatory factors such as (D) IL6, (E) IL1β, and (F) TNFα in mouse lung tissue.

[0026] Figure 8 :Ki67 histochemical staining results of mouse lung tissue. (A) Control group; (B) PPE-induced emphysema group; (C) Emphysema + Luc mRNA LNP treatment group; (D) Emphysema + HGF mRNA LNP treatment group; (E) Emphysema + HGF recombinant protein treatment group; (F) Semi-quantitative statistical results of Ki67 positive cell percentage.

[0027] Fig. 9 :TUNEL staining results of mouse lung tissue. (A) Control group; (B) PPE-induced emphysema group; (C) Emphysema + Luc mRNA LNP treatment group; (D) Emphysema + HGF mRNA LNP treatment group; (E) Emphysema + HGF recombinant protein treatment group. DETAILED DESCRIPTION

[0028] The preferred embodiments of the present invention are described below in conjunction with the accompanying drawings. It should be understood that the preferred embodiments described herein are only used to illustrate and explain the present invention, and are not used to limit the present invention.

[0029] Example 1 Preparation of HGF mRNA lipid nanoparticles (LNP)

[0030] 1. Plasmid Preparation

[0031] This scheme uses the HGF plasmid designed by the research team and the commercially available luciferase plasmid to artificially synthesize the ORF sequence (SEQ ID NO.1) and luciferase sequence (SEQ ID NO.2) encoding the same human HGF natural protein. A DNA plasmid (SEQ ID NO.5) containing the ORF sequence and its upstream and downstream 5'UTR and 3'UTR regulatory sequences (SEQ ID NO.3 and SEQ ID NO.4, respectively) is constructed, amplified and extracted by conventional molecular cloning methods.

[0032] 2. Plasmid DNA restriction enzyme digestion

[0033] The HGF plasmid was digested with restriction endonucleases according to the following digestion system (Table 1):

[0034] Table 1 Reagents used for HGF plasmid digestion

[0035] name Volume (μl) SspI 1μl BnS 1μl Buffer 5μl Plasmid DNA 25 μg Nuclease-free water Make up to a total volume of 50 μl

[0036] Refer to the following enzyme digestion system to perform restriction endonuclease digestion on the luciferase plasmid (Table 2):

[0037] Table 2 Reagents used for luciferase plasmid digestion

[0038] name Volume (μl) BnS 1μl Buffer 5μl Plasmid DNA 25 μg Nuclease-free water Make up to a total volume of 50 μl

[0039] Add the above reagents to the EP tube, mix well, and perform enzyme digestion reaction according to Table 3:

[0040] Table 3 Enzyme digestion reaction system

[0041] temperature time 37℃ 4h 80℃ 20min 4℃ ∞

[0042] 3. In vitro Transcription

[0043] In vitro transcription was performed using the Vazyme in vitro transcription kit, catalog number: DD4202.

[0044] (1) Vortex and mix all components except T7 RNA Polymerase Mix, centrifuge briefly, collect the components at the bottom of the tube, and store on ice until needed.

[0045] (2) Add the following components in sequence as shown in Table 4:

[0046] Table 4 In vitro transcription reaction system

[0047] Components volume T7RNAPolymeraseMix 2μl 10×TranscriptionBuffer 2μl N1-Me-PseudoUTP 1.5μl ATPSolution 1.5μl CTPSolution 1.5μl GTPSolution 1.5μl CAGTrimer 1μl Template DNA 1 μg Nuclease-free water Make up to 20 μl

[0048] (3) Mix the components gently with a pipette, collect by brief centrifugation, and incubate at 37°C for 2 h.

[0049] (4) Add 1 μl of DNase I to the reaction system and incubate at 37°C for 15 min to digest the transcribed DNA template.

[0050] 4. mRNA Purification

[0051] Use HieffNGS RNACleaner magnetic beads (Cat. No.: 12602ES08) to purify the in vitro transcribed mRNA in the previous step:

[0052] (1) Remove RNAcleanbeads from 4°C and equilibrate to room temperature (about 30 min).

[0053] (2) In a Nuclease-free PCR tube, dilute 0.5-5 μg of total RNA to 50 μl with Nuclease-free water. (3) Invert or vortex to mix the magnetic beads thoroughly, pipette 2× magnetic beads (100 μl) into the total RNA sample (50 μl), and mix thoroughly by pipetting 6 times.

[0054] (4) Incubate at room temperature for 5 min to allow the RNA to bind to the magnetic beads.

[0055] (5) Place the sample on a magnetic rack for 5 min. After the solution becomes clear, carefully remove the supernatant.

[0056] (6) Keep the sample on the magnetic rack, add 200 μl of freshly prepared 80% ethanol to rinse the magnetic beads, incubate at room temperature for 30 seconds, and carefully remove the supernatant.

[0057] (7) Repeat step 6 and rinse twice in total.

[0058] (8) Keep the sample on the magnetic rack and open the lid to air-dry the magnetic beads for 5 minutes.

[0059] (9) Remove the sample from the magnetic rack, add 12 μl of Nuclease-free water, pipette up and down 6 times to mix thoroughly, and let stand at room temperature for 5 min.

[0060] (10) Place the sample on a magnetic rack for 5 min. After the solution is clarified, carefully transfer 10 μl of the supernatant to a new Nuclease-free PCR tube to obtain the prepared HGF mRNA.

[0061] 5. Preparation of HGF mRNA LNP and luciferase mRNA LNP

[0062] (5) Preparation of lipid mixture

[0063] Prepare lipidmix according to the system in Table 5 below:

[0064] Table 5 Lipidmix formulation system

[0065] Components volume SM102 (Article No.: O02010) 5.88μl Cholesterol (Product No.: O02003) 18.56μl DMG-PEG (Cat. No.: O02005) 4.68μl DSPC(Article No.:S01005) 9.84μl

[0066] The above is a lipidmix preparation system with a total system of 38.96 μl. During actual operation, it can be proportionally enlarged or reduced according to actual conditions.

[0067] (6) Preparation of HGF mRNA LNP and luciferase mRNA LNP

[0068] Prepare HGF mRNA LNP and luciferase mRNA LNP according to the system in Table 6 below:

[0069] Table 6 HGF / luciferase mRNA LNP formulation system

[0070] Components volume lipidmix 20.16μl EtOH 1.44μl HGF / luciferase mRNA 9.6μl Citrate buffer (10 mM, pH 4) 55.2μl

[0071] The above is a HGF / luciferase mRNA LNP preparation system with a total system of 86.4 μl. In actual operation, it can be proportionally enlarged or reduced according to the actual situation. When preparing the system in Table 5, first use Citrate buffer to dilute the mRNA, then add it to the mixed lipid of lipid mix and EtOH, and use a pipette to quickly blow and mix to prepare HGF / luciferase mRNA LNP.

[0072] Example 2: Verification of the effect of HGF mRNA LNP pulmonary delivery in the treatment of emphysema

[0073] 1. SM102 LNP can specifically target the lungs to deliver mRNA

[0074] In order to screen out the best LNP that can specifically deliver mRNA to the lungs, we used the purchased MC3, SM102, and A LC0315 LNPs to encapsulate 30 μg of luciferase mRNA. After targeted delivery to mice via the airway for 12 hours, we detected the fluorescence expression of organs such as the heart, lungs, liver, kidneys, and spleen ( Figure 1A). The results showed that all three LNPs could successfully deliver luciferase mRNA to the lungs ( Figure 1 B), SM102 performed best, with the smallest difference in fluorescence signal values ​​within the group, and the fluorescence signal only appeared in lung tissue, indicating that SM102 can specifically target the lungs to deliver mRNA ( Figure 1 C). Therefore, we next used SM102 LNP as the optimal vehicle for delivering mRNA to the lung.

[0075] 2. SM102 LNP-encapsulated luciferase mRNA can be stably expressed in the lungs

[0076] To further explore the duration of sustained expression of mRNA delivered by SM102 LNP in the lungs, we used SM102 LNP to encapsulate 30 μg of luciferase mRNA and then delivered it to the lungs via the airway ( Figure 2 A), the fluorescence signals in lung tissue were detected at 6h, 12h, 24h and 48h ( Figure 2 B). The results showed that the fluorescence signal in the lungs increased significantly from 6 hours onwards, continued until 24 hours later, and then began to decline. After 48 hours, the fluorescence signal had decayed to half, indicating that the luciferase mRNA encapsulated by SM102 LNP can be stably present in the lung tissue for at least 24 hours ( Figure 2 C).

[0077] 3. SM102 LNP encapsulated 15 μg mRNA can be significantly enriched in the lungs

[0078] To explore the optimal dose of SM102 LNP loaded with mRNA, we used SM102 LNP to encapsulate different doses of luciferase mRNA (5μg, 15μg, 30μg) and then delivered it to the lungs through the airway ( Figure 3 A), 12 hours later, the fluorescence signal of the lungs was detected by bioluminescence imaging ( Figure 3 B). The results showed that a 15 μg dose of luciferase mRNA could significantly increase the fluorescence signal value in the lungs ( Figure 3 C).

[0079] 4. HGF mRNA-SM102 LNP can promote the repair of lung tissue structure in mice with emphysema

[0080] To investigate the therapeutic effect of HGF mRNA-SM102 LNP on emphysema mice, we instilled HGF mRNA-SM102 LNP into the lungs of emphysema mice through the airway (instillation was performed in the last week of emphysema modeling, once every three days, for a total of two instillations, 50 μl of SM102 LNP containing 20 μg HGF mRNA was instilled into each mouse each time), and instillation of luciferase mRNA-SM102 LNP and HGF recombinant protein was used as controls. The results of mouse lung function tests showed that compared with the control group mice, the dynamic elasticity of the respiratory system of emphysema mice ( Figure 4 A) and tissue elasticity ( Figure 4 D) decreased significantly, and the dynamic compliance of the respiratory system ( Figure 4 B) and quasi-static compliance ( Figure 4 C) was significantly increased. In addition, the forced inspiratory volume ( Figure 4 E) and total lung capacity ( Figure 4 F) also increased significantly. After airway instillation of HGF mRNA-SM102 LNP, the lung function indicators of emphysematous mice, such as dynamic elasticity of the respiratory system, tissue elasticity, dynamic compliance of the respiratory system, quasi-static compliance, forced inspiratory volume, and total lung volume, all recovered, and the therapeutic effect was better than that of HGF recombinant protein. Therefore, HGF mRNA-SM102 LNP can significantly improve the lung function of emphysematous mice. Further HE staining of lung tissue sections showed that the alveolar cavity of emphysematous mice was significantly enlarged ( Figure 5 AE), which was manifested by a significant increase in the mean linear intercept of the alveoli ( Figure 5 F), while HGF mRNA-SM102 LNP can partially repair the abnormally altered alveolar structure. The results of blood routine and blood biochemistry of mice showed that airway instillation of LNP mRNA had no significant effect on the systemic functional status of mice ( Figure 6 ).

[0081] 5. HGF mRNA-SM102 LNP can inhibit lung inflammation, promote alveolar epithelial proliferation and inhibit apoptosis in mice with emphysema

[0082] Previous literature reports that HGF, as a growth factor, has the effects of inhibiting inflammation, promoting cell proliferation, and inhibiting cell apoptosis. To evaluate the improvement of the phenotype of emphysematous mice by HGF mRNA LNP, we further evaluated the changes in lung inflammation, alveolar epithelial cell proliferation, and apoptosis in emphysematous mice. RT-qPCR and ELISA results showed that the expression of inflammatory factors IL6, IL1β, and TNFα in the lung tissue of emphysematous mice was significantly increased, while the expression of inflammatory factors was significantly inhibited after delivering HGF mRNA LNP to the lungs ( Figure 7The results of Ki67 histochemical staining showed that the proliferation of alveolar epithelial cells in mice with emphysema was weakened, while the delivery of HGF mRNA LNP to the lungs significantly promoted the proliferation of alveolar epithelial cells ( Figure 8 ). TUNEL staining results showed that apoptosis of alveolar epithelial cells increased in mice with emphysema, while delivery of HGF mRNA LNP to the lungs significantly inhibited apoptosis of alveolar epithelial cells ( Fig. 9 ).

[0083] In summary, delivery of HGF mRNA LNP to the lungs can significantly inhibit lung tissue inflammation, promote alveolar epithelial cell proliferation, and inhibit alveolar epithelial cell apoptosis in emphysema mice, thereby promoting the repair of lung tissue.

[0084] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. A use of HGF mRNA LNP in the preparation of a drug for treating emphysema, characterized in that: The HGF mRNA LNP contains the HGF mRNA sequence as shown in SEQ ID NO.1, and the HGF mRNA LNP is prepared by the following method: (1) Preparing a lipid mix: the lipid mix includes SM102, cholesterol, DMG-PEG, and DSPC; (2) diluting the HGF mRNA with a citrate buffer, adding it to a lipid mixture of lipidmix and ethanol (EtOH), and quickly pipetting and mixing it to obtain HGF mRNA LNP; Wherein, the volume ratio of SM102, Cholesterol, DMG-PEG and DSPC is 5.88:18.56:4.68:9.

84.

2. The use according to claim 1, characterized in that The volume ratio of lipid mix, EtOH, HGF mRNA, and Citrate buffer is 20.16:1.44:9.6:55.2, among which the concentration of Citrate buffer is 10mM, pH=4.

3. The use according to claim 1, characterized in that The treatment includes inhibiting emphysematous lung inflammation, promoting alveolar epithelial proliferation and inhibiting apoptosis thereof.

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