Preparation and clinical application of growth differentiation factor 11 (GDF11) mRNA for promoting wound healing

Abstract

The invention belongs to the field of clinical technology, and provides preparation and clinical application of growth differentiation factor 11 (GDF11) mRNA for promoting wound healing, and the preparation method comprises the following steps: S1, carrying out N1 psi modification on GDF11 mRNA, and replacing uracil in an original sequence with N1 psi; s2, synthesizing the modified GDF11 mRNA (messenger Ribonucleic Acid); s3, purifying the synthesized GDF11 mRNA (messenger Ribonucleic Acid), so as to obtain mRNA with modified nucleoside; by optimizing the preparation method of growth differentiation factor 11 (GDF11) mRNA, the preparation method comprises the following steps: performing N1 psi modification, synthesizing by adopting a vaccinia enzyme m capping 7G capping system, adding a poly (A) tail, purifying, and wrapping in lipid nanoparticles (LNP) to form a stable delivery system, so that the stability and translation efficiency of mRNA are remarkably improved, and the stability and translation efficiency of the GDF11 are remarkably improved. And the defects existing in treatment by directly using the GDF11 protein in the prior art are overcome, an efficient and safe new strategy is provided for diabetes wound treatment, and remarkable beneficial effects are achieved.

Description

Technical Field

[0001] The present invention belongs to the field of clinical technology, and specifically relates to the preparation and clinical application of growth differentiation factor 11 (GDF11) mRNA for promoting wound healing. Background Art

[0002] Clinically, skin defects are common and caused by various factors. In the field of trauma, the repair and functional reconstruction of skin wounds have become an important problem to be solved urgently. Diabetic foot ulcer is a common and dangerous complication of diabetes, caused by the oxidation of glucose in diabetic wounds, which damages the healing response, presents as chronic non-healing wounds, and may ultimately lead to lower limb amputation or death. Diabetic patients have a high risk of developing diabetic foot ulcers, which bring great pain and economic burden to patients. Therefore, there is an urgent need for effective treatment strategies for diabetic wounds.

[0003] Growth differentiation factor 11 (GDF-11), also known as bone morphogenetic protein 11 (BMP-11), is a secreted protein belonging to the transforming growth factor-β (TGF-β) superfamily and has a wide range of functions in cell aggregation, migration, adhesion, differentiation, development, angiogenesis, neurotissue protection and repair. Existing studies have shown that GDF11 can promote new blood vessel formation by stimulating the mobilization, migration and homing of endothelial progenitor cells (EPCs) to the injured area, thereby enhancing the regeneration of skin tissue and effectively treating diabetic wounds. However, the direct use of GDF11 protein for treatment has disadvantages such as poor absorption of protein macromolecules, short half-life, complex purification process, and high production cost, which limit its clinical application.

[0004] Gene-based drugs, such as mRNA, as potential alternatives to recombinant protein therapy, have many advantages. The main principle of mRNA drugs is to produce functional proteins from cells in the body without directly injecting protein drugs. The process of synthesizing mRNA by in vitro transcription (IVT) is relatively simple and low-cost, and can be quickly applied to various treatments. In addition, mRNA drugs can initiate translation without entering the cell nucleus and will not insert into the genome, having higher safety.

[0005] Therefore, those skilled in the art have proposed the preparation and clinical application of growth differentiation factor 11 (GDF11) mRNA for promoting wound healing to solve the problems raised in the background art. Summary of the Invention

[0006] To solve the above technical problems, the present invention provides the preparation and clinical application of growth differentiation factor 11 (GDF11) mRNA for promoting wound healing, so as to solve the problems that the direct use of GDF11 protein for treatment in the prior art has disadvantages such as poor absorption of protein macromolecules, short half-life, complex purification process, high production cost, etc., which limit its clinical application.

[0007] In a first aspect, the present invention provides a method for preparing GDF11 mRNA for promoting wound healing and its application, which specifically includes the following steps:

[0008] S1. Modify GDF11 mRNA: Perform N1Ψ modification on GDF11 mRNA, replacing uracil in the original sequence with N1Ψ to improve the stability and translation efficiency of mRNA;

[0009] S2. Synthesize the modified GDF11 mRNA: Use the vaccinia enzyme m capping 7G capping system for synthesis, and add a poly(A) tail after synthesis to enhance the stability and expression efficiency of mRNA;

[0010] S3. Purify GDF11 mRNA: Purify the synthesized GDF11 mRNA to obtain highly purified modified mRNA;

[0011] S4. Prepare the GDF11 mRNA-LNP composition: Wrap the modified and purified GDF11 mRNA in lipid nanoparticles (LNP) to form a stable delivery system.

[0012] In a second aspect, the present invention also provides the application of the GDF11 mRNA prepared by the above preparation method in the preparation of a pharmaceutical composition for promoting wound healing, especially for treating diabetic wounds.

[0013] Preferably, the pharmaceutical composition contains GDF11 mRNA and a pharmaceutically acceptable carrier or excipient.

[0014] Preferably, GDF11 mRNA is encapsulated in LNP.

[0015] Compared with the prior art, the present invention has the following beneficial effects:

[0016] By optimizing the preparation method of growth differentiation factor 11 (GDF11) mRNA, including performing N1Ψ modification, using the vaccinia enzyme m capping 7G capping system for synthesis and adding a poly(A) tail, purification, and encapsulation in lipid nanoparticles (LNP) to form a stable delivery system, the present invention significantly improves the stability and translation efficiency of mRNA, and solves the defects existing in the direct use of GDF11 protein for treatment in the prior art, providing an efficient and safe new strategy for the treatment of diabetic wounds, with significant beneficial effects. Description of the Drawings

[0017] Figure 1 It is a comparison chart of blood glucose values of diabetic model mice and normal mice of the present invention;

[0018] Figure 2Schematic diagram of the effect of GDF11 mRNA-LNP of the present invention on wound healing in a mouse diabetic wound model;

[0019] Figure 3 Schematic diagram of the effect of GDF11 mRNA-LNP of the present invention on the wound healing rate in a mouse diabetic wound model;

[0020] Figure 4 Schematic diagram of the GDF11 gene sequence of the present invention. Detailed implementation manners

[0021] The following further describes in detail the implementation manners of the present invention in conjunction with the drawings and examples. The following examples are used to illustrate the present invention, but cannot be used to limit the scope of the present invention.

[0022] Example: The present invention provides a preparation of growth differentiation factor 11 (GDF11) mRNA for promoting wound healing;

[0023] 1. This method includes three steps: modification, synthesis, and purification:

[0024] 1) Modification of GDF11 mRNA

[0025] Replace uracil (thymine in eukaryotes) in the original GDF11 sequence with N 1 ψ-modified nucleosides to improve the stability of mRNA and reduce immunogenicity.

[0026] 2) Synthesis of GDF11 mRNA

[0027] Based on the modified GDF11 mRNA sequence, in vitro transcription (IVT) synthesis is carried out. The reaction system contains all four nucleoside triphosphates (natural and modified), all administered at a final concentration of 1.8 mM, specifically 5'-methylcytosine (5mC), N 1 -methylpseudouridine (N 1 mΨ), 5'-methyluracil (5mU), and N 1 -methylpseudouridine (or 5mC and Ψ, selected according to specific optimization results), as Figure 4 shown.

[0028] Use the vaccinia enzyme m capping 7G capping system (New England Biolabs) to perform 5'-end capping on the mRNA.

[0029] Add a poly(A) tail to the 3' end of the mRNA to improve its stability and translation efficiency in cells. The specific operation is as follows: The selected mRNA is tailed with poly(A) in a reaction containing approximately 1.5 μg / μL RNA, 5 mmol / L adenosine triphosphate, and 60 U / μL yeast poly(A) polymerase (USB, Cleveland, OH), and incubated at 30 °C for 3 hours. The length of the poly(A) tail is estimated to be approximately 200 nucleotides long.

[0030] 3) Purification of GDF11 mRNA

[0031] Remove the DNA template by digestion with Turbo DNase (Ambion, Austin, TX).

[0032] Purify the transcript using LiCl precipitation and 75% ethanol washing.

[0033] Determine the concentration of the RNA reconstituted in water by measuring the optical density at 260 nm.

[0034] Prove the efficient incorporation of modified nucleotides into the transcript by HPLC analysis.

[0035] All RNA samples were analyzed by denaturing agarose gel electrophoresis to ensure quality.

[0036] 2. Establishment of a diabetic mouse model

[0037] 1) Induction of a mouse diabetic model

[0038] Take 60 mice and fast them for 12 hours without water deprivation.

[0039] Inject a solution of streptozotocin (STZ) (10 mg / ml) intraperitoneally at a dose of 1.8 mg STZ / 10 g body weight of the mouse.

[0040] After injecting STZ, immediately give 5% glucose water for 12 hours to relieve the acute toxic effect of STZ.

[0041] Resume normal feeding and drinking the next day.

[0042] Measure the postprandial blood glucose level 7 days later. As Figure 1 shown, mice with a blood glucose concentration higher than 16.7 mmol / L are considered diabetic mice (DM), indicating successful model establishment.

[0043] 3. Establishment of a diabetic wound model and grouped drug administration

[0044] 1) Establishment of a diabetic wound model

[0045] Thirty diabetic model mice and ten normal mice were used as negative controls.

[0046] Anesthetize the mice by intraperitoneal injection of avertin solution (0.02 g / ml) according to the standard of 0.10 ml / 10 g body weight of the mice.

[0047] Depilate the back to expose the skin, and use a skin punch to create a circular full-thickness skin wound with a diameter of 8 mm at the lower 1 / 3 of the mouse back (including epidermis, dermis and subcutaneous tissue).

[0048] Each mouse was housed individually in a cage to avoid interference and infection with each other.

[0049] 2) Grouping and administration

[0050] The thirty diabetic wound model mice were randomly divided into three groups:

[0051] Blank control group (DM): Drop 0.1% BSA buffer on the wound.

[0052] Positive control group (GDF11+DM): Apply 50 ng / ml of recombinant GDF11 protein (rGDF11) on the wound.

[0053] Experimental group (GDF11mRNA-LNP+DM): Intradermally inject 50 ng / ml of GDF11mRNA-LNP solution into the wound.

[0054] In addition, ten non-diabetic normal wound model mice were used as negative control group (ctl), and the treatment method was the same as that of the experimental group, but without diabetes induction and GDF11-related treatment.

[0055] 3) Administration method

[0056] The positive control group and the experimental group were administered once every two days, and the administration time points were fixed.

[0057] The total volume of each administration was 50 μl.

[0058] The administration method of the positive control group was application, and the administration method of the experimental group was intradermal injection.

[0059] 0.1% BSA buffer was dropped on the wound of the blank control group, and the administration frequency and volume were the same as those of the experimental group.

[0060] 4. Statistics and analysis of wound healing in model mice

[0061] 1) Recording of wound healing

[0062] Record the area of each wound once a day using a single-lens reflex camera, and the recording time is fixed every day.

[0063] Keep the distance between the lens and the wound consistent (about 30 cm) to ensure the accuracy of the recording.

[0064] 2) Calculation of wound healing rate

[0065] Integrate the wound area using ImageG software.

[0066] The wound healing rate is calculated using the following formula: Wound healing rate = (Initial wound area - Final wound area) / Initial wound area (for the same wound on the same mouse).

[0067] 3) Statistical analysis

[0068] Perform statistical analysis on the wound healing of mice in each group to compare the differences between different treatment groups.

[0069] The results are as Figure 2 and Figure 3 shown. The wound healing rate of the experimental group (GDF11mRNA-LNP + DM) is higher than that of the positive control group (GDF11 + DM) and the blank control group (DM), indicating that GDF11mRNA-LNP has a significant pharmacological effect in promoting diabetic wound healing.

[0070] As can be seen from the above, the present invention has the following beneficial effects:

[0071] First, the staff will perform N1Ψ modification on GDF11mRNA, replacing uracil in the original sequence with N1Ψ to improve the stability and translation efficiency of mRNA. This modification can enhance the stability of mRNA in cells, reduce the risk of its degradation, and thus ensure the continuous and efficient expression of GDF11 protein.

[0072] Next, use the vaccinia enzyme m capping 7G capping system for synthesis and add a poly(A) tail after synthesis to further enhance the stability and expression efficiency of mRNA. The optimization of this step makes the mRNA synthesis process more efficient and improves its translation activity in cells.

[0073] Then, purify the synthesized GDF11mRNA to obtain highly purified modified mRNA. The introduction of the purification step ensures the quality and purity of the final product, reducing the potential risks of immunogenicity and toxicity.

[0074] In addition, the present invention also encapsulates the modified and purified GDF11mRNA in lipid nanoparticles (LNP) to form a stable delivery system. As a carrier, LNP can protect mRNA from being damaged by the in vivo environment and at the same time promote its effective delivery to target cells to achieve precise treatment.

[0075] Through the above technical solution, the present invention solves the disadvantages of poor absorption of large protein molecules, short half-life, complex purification process, and high production cost in the prior art when directly using GDF11 protein for treatment. Compared with directly using GDF11 protein, the GDF11 mRNA drug provided by the present invention has higher stability, lower immunogenicity, and better therapeutic effects.

[0076] Specifically, the GDF11 mRNA drug prepared by the present invention shows significant efficacy in the treatment of diabetic wounds. The experimental results show that the wound healing rate of the diabetic wound model mice treated with GDF11 mRNA-LNP is significantly higher than that of the control group treated with recombinant GDF11 protein and the blank control group. This indicates that the GDF11 mRNA drug provided by the present invention has a significant pharmacological effect of promoting the healing of diabetic wounds, providing a new effective strategy for the treatment of diabetic wounds.

[0077] In summary, by optimizing the preparation method and delivery system of GDF11 mRNA, the present invention realizes its efficient application in the treatment of diabetic wounds, having significant technical advantages and clinical application prospects.

[0078] The embodiments of the present invention are given for the purpose of illustration and description. Although the embodiments of the present invention have been shown and described above, it can be understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those of ordinary skill in the art can make changes, modifications, substitutions, and variations to the above embodiments within the scope of the present invention.

Claims

1. A method for preparing growth differentiation factor 11 (GDF11) mRNA for promoting wound healing, characterized in that: The method steps include: S1, N1Ψ modification of GDF11 mRNA, replacing uracil in the original sequence with N1Ψ; S2, synthesis of modified GDF11 mRNA; S3. Purify the synthesized GDF11 mRNA to obtain mRNA with modified nucleosides.

2. The preparation of a growth differentiation factor 11 (GDF11) mRNA for promoting wound healing according to claim 1, characterized in that: The synthesis in step S2 uses the vaccinia enzyme m capping 7G capping system, and a poly tail is added after synthesis.

3. The preparation of a growth differentiation factor 11 (GDF11) mRNA for promoting wound healing according to claim 1, characterized in that: The method also includes encapsulating the modified and purified GDF11 mRNA in lipid nanoparticles to form a GDF11 mRNA-LNP composition.

4. Use of the growth differentiation factor 11 mRNA prepared by the preparation method according to any one of claims 1 to 3 in preparing a pharmaceutical composition for promoting wound healing.

5. The use according to claim 4, characterized in that: The pharmaceutical composition is used for treating diabetic wounds.

6. A pharmaceutical composition for promoting wound healing, characterized in that: The invention comprises growth differentiation factor 11 mRNA prepared by the preparation method according to any one of claims 1 to 3 and a pharmaceutically acceptable carrier or excipient.

7. The pharmaceutical composition according to claim 6, characterized in that The pharmaceutical composition further comprises lipid nanoparticles, and the GDF11 mRNA is encapsulated in the LNPs.

Images