Use of skin reprogramming factor formulation in preparation of drug for promoting skin wound healing

Abstract

Provided is a use of a skin reprogramming factor formulation in the preparation of a drug for promoting skin wound healing. The skin reprogramming factor formulation comprises 2-4 mole parts of an mRNA nucleic acid molecule encoding an Oct4 transcription factor, 1 mole part of an mRNA nucleic acid molecule encoding a Sox2 transcription factor, 1 mole part of an mRNA nucleic acid molecule encoding a Klf4 transcription factor, 1 mole part of an mRNA nucleic acid molecule encoding a Glis1 transcription factor, and 1 mole part of an mRNA nucleic acid molecule encoding a Lin28 transcription factor. After the skin reprogramming factor formulation is introduced into a skin wound, various reprogramming factors are generated by means of skin cells on the wound, the reprogramming factors can be expressed at full-thickness skin to efficiently repair damaged tissue, and the reprogramming factors can quickly repair the skin wound and reduce scars.

Description

Use of skin reprogramming factor preparations in the preparation of drugs promoting skin wound healing Technical Field

[0001] The present application relates to the field of biomedicine technology, and in particular to the use of a skin reprogramming factor preparation in the preparation of a drug promoting skin wound healing. Background Art

[0002] The skin is the largest organ in the human body, and wounds are almost inevitable. Skin repair is a complex biological process involving the interactions of multiple cell types, signaling molecules, and the extracellular matrix. Rapid and high-quality repair of skin wounds is a significant unmet clinical need. Skin aging is an independent risk factor for delayed wound healing, particularly in elderly individuals with pressure sores or diabetic foot. Slow healing, or even inability to repair, of skin wounds can become life-threatening. Scarring is also a significant clinical challenge in skin wound repair.

[0003] Currently, since macrophages and fibroblasts that accumulate in wounds can secrete epidermal growth factor (EGF), which stimulates the proliferation and migration of epithelial cells and mediates the differentiation of keratinocytes into epithelial cells, this mechanism suggests that EGF can promote wound epithelialization and rebuild the epithelial barrier. Existing technologies use EGF for wound healing, but its effectiveness in promoting rapid repair of skin wounds and reducing scarring needs to be improved.

[0004] Summary of the Invention

[0005] In view of the above problems, the present invention provides an embodiment of a skin reprogramming factor preparation for use in the preparation of a drug for promoting skin wound healing, in order to solve the above technical problems.

[0006] In a first aspect, an embodiment of the present application provides a use of a skin reprogramming factor preparation in the preparation of a drug that promotes skin wound healing, wherein the skin reprogramming factor preparation comprises 2 to 4 molar parts of an mRNA nucleic acid molecule encoding an Oct4 transcription factor, 1 molar part of an mRNA nucleic acid molecule encoding a Sox2 transcription factor, 1 molar part of an mRNA nucleic acid molecule encoding a Klf4 transcription factor, 1 molar part of an mRNA nucleic acid molecule encoding a Glis1 transcription factor, and 1 molar part of an mRNA nucleic acid molecule encoding a Lin28 transcription factor.

[0007] Optionally, the skin reprogramming factor preparation includes 3 molar parts of an mRNA nucleic acid molecule encoding an Oct4 transcription factor, 1 molar part of an mRNA nucleic acid molecule encoding a Sox2 transcription factor, 1 molar part of an mRNA nucleic acid molecule encoding a Klf4 transcription factor, 1 molar part of an mRNA nucleic acid molecule encoding a Glis1 transcription factor, and 1 molar part of an mRNA nucleic acid molecule encoding a Lin28 transcription factor.

[0008] Optionally, the skin reprogramming factor preparation further comprises a first solvent, the concentration of citrate in the first solvent is 8 to 12 mmol / L, and the concentration of sodium chloride in the first solvent is 120 to 140 mmol / L.

[0009] Optionally, the concentration of citrate in the first solvent is 10 mmol / L, the concentration of sodium chloride in the first solvent is 130 mmol / L, and the pH value of the first solvent is 7.5.

[0010] Optionally, each of the mRNA nucleic acid molecules includes a 5' cap structure, a 5' UTR sequence, an mRNA coding sequence of a corresponding transcription factor, a 3' UTR sequence and a polyadenylation sequence.

[0011] Optionally, some or all of the nucleotides in the mRNA nucleic acid molecule encoding the Oct4 transcription factor, the mRNA nucleic acid molecule encoding the Sox2 transcription factor, the mRNA nucleic acid molecule encoding the Klf4 transcription factor, the mRNA nucleic acid molecule encoding the Glis1 transcription factor, and the mRNA nucleic acid molecule encoding the Lin28 transcription factor are chemically modified to improve the stability of the mRNA nucleic acid molecules in vivo.

[0012] Optionally, the chemical modification includes replacing 100% of the cytosine in the mRNA nucleic acid molecule encoding the Oct4 transcription factor, the mRNA nucleic acid molecule encoding the Sox2 transcription factor, the mRNA nucleic acid molecule encoding the Klf4 transcription factor, the mRNA nucleic acid molecule encoding the Glis1 transcription factor, and the mRNA nucleic acid molecule encoding the Lin28 transcription factor with 5-methylcytosine, and replacing 100% of the uracil in the mRNA nucleic acid molecule encoding the Oct4 transcription factor, the mRNA nucleic acid molecule encoding the Sox2 transcription factor, the mRNA nucleic acid molecule encoding the Klf4 transcription factor, the mRNA nucleic acid molecule encoding the Glis1 transcription factor, and the mRNA nucleic acid molecule encoding the Lin28 transcription factor with N1-methylpseudouridine.

[0013] Optionally, the DNA sequence of the Oct4 transcription factor is shown as SEQ ID NO.3, the DNA sequence of the Sox2 transcription factor is shown as SEQ ID NO.4, the DNA sequence encoding the Klf4 transcription factor is shown as SEQ ID NO.5, the DNA sequence of the Glis1 transcription factor is shown as SEQ ID NO.6, and the DNA sequence of the Lin28 transcription factor is shown as SEQ ID NO.7.

[0014] Optionally, the DNA sequence of the 5'UTR sequence is shown as SEQ ID NO.1, and the DNA sequence of the 3'UTR sequence is shown as SEQ ID NO.2.

[0015] Optionally, the skin reprogramming factor preparation is an injectable preparation.

[0016] The skin reprogramming factor preparation provided in the embodiments of the present application is used in the preparation of a drug for promoting skin wound healing. The skin reprogramming factor preparation includes 2 to 4 molar parts of an mRNA nucleic acid molecule encoding an Oct4 transcription factor, 1 molar part of an mRNA nucleic acid molecule encoding a Sox2 transcription factor, 1 molar part of an mRNA nucleic acid molecule encoding a Klf4 transcription factor, 1 molar part of an mRNA nucleic acid molecule encoding a Glis1 transcription factor, and 1 molar part of an mRNA nucleic acid molecule encoding a Lin28 transcription factor. After the above-mentioned skin reprogramming factor preparation is introduced into a skin wound, each reprogramming factor is produced by the skin cells on the wound. Each reprogramming factor can be expressed in the entire layer of the skin, efficiently repairing damaged tissue. Each reprogramming factor can quickly repair the skin wound and reduce scars.

[0017] These and other aspects of the present application will become more readily apparent from the description of the following embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] FIG1 shows an inverted microscope image of cell proliferation and migration of human keratinocytes in a scratch test in Example 2 of the present application.

[0019] FIG2 shows a quantitative bar graph of the cell proliferation and migration results of the human keratinocyte scratch test in Example 2 of the present application.

[0020] FIG3 shows an inverted microscope image of cell proliferation and migration of human fibroblasts in a scratch test according to Example 3 of the present application.

[0021] FIG4 shows a quantitative bar graph of the cell proliferation and migration results of the human fibroblast scratch assay in Example 3 of the present application.

[0022] FIG5 shows the distribution diagram of fluorescent protein expression in the fluorescent expression experiment of the mouse skin defect model in Example 4 of the present application.

[0023] FIG6 shows a comparison of wound surface changes in the wound healing experiment of the mouse skin defect model in Example 5 of the present application.

[0024] FIG7 shows a quantitative graph of wound healing quality in the wound healing experiment of the mouse skin defect model in Example 5 of the present application.

[0025] FIG8 shows a quantitative graph of the wound healing quality assessment results in the mouse skin defect model wound healing experiment of Example 5 of the present application.

[0026] FIG9 shows the results of a skin wound healing experiment using skin reprogramming factor preparations in different ratios according to Example 6 of the present application. DETAILED DESCRIPTION

[0027] The embodiments of the present application are described in detail below. Examples of the embodiments are shown in the accompanying drawings, wherein the same or similar reference numerals throughout represent the same or similar elements or elements having the same or similar functions. The embodiments described below with reference to the accompanying drawings are exemplary and are only used to explain the present application and are not to be construed as limiting the present application.

[0028] In order to enable those skilled in the art to better understand the solutions of the present application, the technical solutions in the embodiments of the present application will be clearly and completely described below in conjunction with the drawings in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, not all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without making creative efforts are within the scope of protection of this application.

[0029] In the embodiments of the present application, it should be noted that, in this document, relational terms such as first and second, etc., are merely used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any actual relationship or order between these entities or operations.

[0030] Moreover, the terms "comprises," "comprising," or any other variations thereof are intended to encompass non-exclusive inclusion, such that a process, method, article, or apparatus that includes a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, method, article, or apparatus. In the absence of further limitations, an element defined by the phrase "comprising a ..." does not preclude the presence of additional identical elements in the process, method, article, or apparatus that includes the element.

[0031] In the description of the embodiments of this application, words such as "example" or "for example" are used to indicate an example, illustration, or description. Any embodiment or design described as "for example" or "for example" in the embodiments of this application is not to be construed as being preferred or having more advantages than another embodiment or design. The use of words such as "example" or "for example" is intended to clearly present relative concepts.

[0032] In addition, in the embodiments of the present application, "plurality" refers to two or more. In view of this, in the embodiments of the present application, "plurality" can also be understood as "at least two". "At least one" can be understood as one or more, for example, one, two, or more. For example, "including at least one" means including one, two, or more, and does not limit which ones are included. For example, "including at least one of A, B, and C" means including A, B, C, A and B, A and C, B and C, or A, B, and C.

[0033] It should be noted that in the embodiments of the present application, "and / or" describes the association relationship between associated objects, indicating that three relationships can exist. For example, A and / or B can represent three situations: A exists alone, A and B exist simultaneously, and B exists alone. In addition, the character " / ", unless otherwise specified, generally indicates that the associated objects are in an "or" relationship.

[0034] It should be noted that in the embodiments of the present application, "connection" can be understood as electrical connection, and the connection between two electrical components can be a direct or indirect connection between the two electrical components. For example, the connection between A and B can be either a direct connection between A and B or an indirect connection between A and B through one or more other electrical components.

[0035] The experimental methods in the following examples are conventional methods unless otherwise specified.

[0036] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0037] One embodiment of the present application provides a skin reprogramming factor preparation, comprising 2 to 4 molar parts of an mRNA nucleic acid molecule encoding an Oct4 transcription factor, 1 molar part of an mRNA nucleic acid molecule encoding a Sox2 transcription factor, 1 molar part of an mRNA nucleic acid molecule encoding a Klf4 transcription factor, 1 molar part of an mRNA nucleic acid molecule encoding a Glis1 transcription factor, and 1 molar part of an mRNA nucleic acid molecule encoding a Lin28 transcription factor.

[0038] In this embodiment, after the skin reprogramming factor preparation is introduced into the skin wound, the mRNA nucleic acid molecule encoding the Oct4 transcription factor, the mRNA nucleic acid molecule encoding the Sox2 transcription factor, the mRNA nucleic acid molecule encoding the Klf4 transcription factor, the mRNA nucleic acid molecule encoding the Glis1 transcription factor, and the mRNA nucleic acid molecule encoding the Lin28 transcription factor are produced by the skin cells on the wound surface to produce various reprogramming factors. Each reprogramming factor can be expressed in the entire layer of the skin, thereby initiating partial epigenetic reprogramming. The cells of the entire layer of skin are epigenetically repaired and readjusted, making the epigenetic state of the cells closer to the youthful state, restoring the mitochondrial function of the cells, improving the cell energy metabolism and increasing oxidative stress, stimulating the proliferation and migration of epithelial cells on the wound surface, participating in the remodeling of the extracellular matrix and regulating the immune response, enhancing the proliferation and regeneration ability of the cells, and more efficiently repairing the damaged tissue, thereby enabling each reprogramming factor to achieve the purpose of rapidly repairing the skin wound.

[0039] As an embodiment, the skin reprogramming factor preparation of this example includes 3 molar parts of mRNA nucleic acid molecules encoding Oct4 transcription factor, 1 molar part of mRNA nucleic acid molecules encoding Sox2 transcription factor, 1 molar part of mRNA nucleic acid molecules encoding Klf4 transcription factor, 1 molar part of mRNA nucleic acid molecules encoding Glis1 transcription factor, and 1 molar part of mRNA nucleic acid molecules encoding Lin28 transcription factor.

[0040] As an embodiment, the skin reprogramming factor preparation of this embodiment further includes a first solvent, in which the mRNA nucleic acid molecule encoding the Oct4 transcription factor, the mRNA nucleic acid molecule encoding the Sox2 transcription factor, the mRNA nucleic acid molecule encoding the Klf4 transcription factor, the mRNA nucleic acid molecule encoding the Glis1 transcription factor, and the mRNA nucleic acid molecule encoding the Lin28 transcription factor are dissolved. The first solvent can be formed by a mixture of citrate and saline, wherein the concentration of citrate in the first solvent is 8 to 12 mmol / L, and the concentration of sodium chloride in the first solvent is 120 to 140 mmol / L.

[0041] In this embodiment, the first solvent can make each mRNA nucleic acid molecule less susceptible to degradation.

[0042] In some embodiments, the concentration of citrate in the first solvent is 10 mmol / L, the concentration of sodium chloride in the first solvent is 130 mmol / L, and the pH value of the first solvent is 7.5.

[0043] As an embodiment, each of the mRNA nucleic acid molecules includes a 5' cap structure, a 5' UTR sequence, an mRNA coding sequence of a corresponding transcription factor, a 3' UTR sequence and a polyadenylation sequence.

[0044] Specifically, the mRNA nucleic acid molecule encoding the Oct4 transcription factor includes a 5' cap structure, a 5' UTR sequence, the mRNA coding sequence of the Oct4 transcription factor, a 3' UTR sequence and a polyadenylic acid sequence; the mRNA nucleic acid molecule encoding the Sox2 transcription factor includes a 5' cap structure, a 5' UTR sequence, the mRNA coding sequence of the Sox2 transcription factor, a 3' UTR sequence and a polyadenylic acid sequence; the mRNA nucleic acid molecule encoding the Klf4 transcription factor includes a 5' cap structure, a 5' UTR sequence, the mRNA coding sequence of the Klf4 transcription factor, a 3' UTR sequence and a polyadenylic acid sequence; the mRNA nucleic acid molecule encoding the Glis1 transcription factor includes a 5' cap structure, a 5' UTR sequence, the mRNA coding sequence of the Glis1 transcription factor, a 3' UTR sequence and a polyadenylic acid sequence; the mRNA nucleic acid molecule encoding the Lin28 transcription factor includes a 5' cap structure, a 5' UTR sequence, the mRNA coding sequence of the Lin28 transcription factor, a 3' UTR sequence and a polyadenylic acid sequence.

[0045] As an embodiment, some or all of the nucleotides in the mRNA nucleic acid molecule encoding the Oct4 transcription factor, the mRNA nucleic acid molecule encoding the Sox2 transcription factor, the mRNA nucleic acid molecule encoding the Klf4 transcription factor, the mRNA nucleic acid molecule encoding the Glis1 transcription factor, and the mRNA nucleic acid molecule encoding the Lin28 transcription factor are chemically modified to improve the stability of the mRNA nucleic acid molecules in vivo.

[0046] In some embodiments, the chemical modification includes replacing 100% of the cytosine in the mRNA nucleic acid molecule encoding the Oct4 transcription factor, the mRNA nucleic acid molecule encoding the Sox2 transcription factor, the mRNA nucleic acid molecule encoding the Klf4 transcription factor, the mRNA nucleic acid molecule encoding the Glis1 transcription factor, and the mRNA nucleic acid molecule encoding the Lin28 transcription factor with 5-methylcytosine, and replacing 100% of the uracil in the mRNA nucleic acid molecule encoding the Oct4 transcription factor, the mRNA nucleic acid molecule encoding the Sox2 transcription factor, the mRNA nucleic acid molecule encoding the Klf4 transcription factor, the mRNA nucleic acid molecule encoding the Glis1 transcription factor, and the mRNA nucleic acid molecule encoding the Lin28 transcription factor with N1-methylpseudouridine.

[0047] In some embodiments, the DNA sequence of the Oct4 transcription factor is shown as SEQ ID NO.3, the DNA sequence of the Sox2 transcription factor is shown as SEQ ID NO.4, the DNA sequence encoding the Klf4 transcription factor is shown as SEQ ID NO.5, the DNA sequence of the Glis1 transcription factor is shown as SEQ ID NO.6, and the DNA sequence of the Lin28 transcription factor is shown as SEQ ID NO.7.

[0048] In some embodiments, the DNA sequence of the 5'UTR sequence is shown as SEQ ID NO.1, and the DNA sequence of the 3'UTR sequence is shown as SEQ ID NO.2.

[0049] In some embodiments, the skin reprogramming factor preparation is an injectable preparation.

[0050] The present application also provides an embodiment of the use of the above-mentioned skin reprogramming factor preparation in the preparation of a drug that promotes skin wound healing.

[0051] In this embodiment, after the above-mentioned skin reprogramming factor preparation is introduced into the skin wound, the skin cells on the wound produce various reprogramming factors, and each reprogramming factor can be expressed in the entire layer of the skin, efficiently repairing damaged tissue. Each reprogramming factor can quickly repair the skin wound and reduce scars.

[0052] Example 1: Preparation of skin reprogramming factor preparation

[0053] The skin reprogramming factor preparation of this embodiment can be prepared according to the following steps:

[0054] S11, Preparation of DNA templates for each transcription factor

[0055] The Oct4 transcription factor DNA template includes a T7 promoter as shown in SEQ ID NO.10, a 5'UTR DNA sequence as shown in SEQ ID NO.1, an Oct4 transcription factor DNA sequence as shown in SEQ ID NO.3, and a 3'UTR DNA sequence as shown in SEQ ID NO.2;

[0056] The Sox2 transcription factor DNA template includes a T7 promoter as shown in SEQ ID NO.10, a 5'UTR DNA sequence as shown in SEQ ID NO.1, a Sox2 transcription factor DNA sequence as shown in SEQ ID NO.4, and a 3'UTR DNA sequence as shown in SEQ ID NO.2;

[0057] The Klf4 transcription factor DNA template includes a T7 promoter as shown in SEQ ID NO.10, a 5'UTR DNA sequence as shown in SEQ ID NO.1, a Klf4 transcription factor DNA sequence as shown in SEQ ID NO.5, and a 3'UTR DNA sequence as shown in SEQ ID NO.2;

[0058] The Glis1 transcription factor DNA template includes a T7 promoter as shown in SEQ ID NO.10, a 5'UTR DNA sequence as shown in SEQ ID NO.1, a Glis1 transcription factor DNA sequence as shown in SEQ ID NO.6, and a 3'UTR DNA sequence as shown in SEQ ID NO.2;

[0059] The Lin28 transcription factor DNA template includes a T7 promoter as shown in SEQ ID NO.10, a 5'UTR DNA sequence as shown in SEQ ID NO.1, a Lin28 transcription factor DNA sequence as shown in SEQ ID NO.7, and a 3'UTR DNA sequence as shown in SEQ ID NO.2;

[0060] The DNA templates of each transcription factor were synthesized according to the above sequence information and can be directly ordered from IDT Corporation in the United States.

[0061] S12, preparation of DNA templates for each transcription factor

[0062] By PCR, poly-(a) tails were added to the Oct4 transcription factor DNA template, Sox2 transcription factor DNA template, Klf4 transcription factor DNA template, Glis1 transcription factor DNA template and Lin28 transcription factor DNA template, respectively, to obtain the Oct4 transcription factor DNA full template, Sox2 transcription factor DNA full template, Klf4 transcription factor DNA full template, Glis1 transcription factor DNA full template and Lin28 transcription factor DNA full template, respectively.

[0063] Prepare PCR premix according to Table 1 (total volume 200 μl, 25 μl for each of eight reactions)

[0064] Table 1. Composition of PCR master mix

[0065] Among them, the tailing primer-F1 is shown as SEQ ID NO.11, and the tailing primer-T120 is shown as SEQ ID NO.12.

[0066] Perform PCR using the conditions in Table 2:

[0067] Table 2. Tailing PCR conditions

[0068] Check the quality of PCR products by gel electrophoresis.

[0069] Gel excision and recovery of PCR products (QIAquick PCR purification kit, Qiagen, cat. no. 28106): The final concentration of the tail template was adjusted to 100 ng / μl and used as the full DNA template for each transcription factor.

[0070] S13, in vitro transcription synthesis of each transcription factor mRNA nucleic acid molecule

[0071] 13-1. Assemble the mRNA cap structure and nucleotide mixture according to Table 3:

[0072] The cap structure N7mGpppGm RNA cap analog (New England Biolabs), Methylcytidine-5′-triphosphate (Me-CTP; Trilink) 100% replaced CTP, N1-methylpseudouridine (Me-Pseudo-UTP; Trilink) 100% replaced UTP; other components were from the MEGAscript T7 kit (Ambion).

[0073] Table 3. mRNA cap structure and nucleotide mixture

[0074] 13-2. Assemble the mRNA in vitro transcription system according to Table 4:

[0075] Table 3. mRNA in vitro transcription system

[0076] 13-3. Place the reaction in a PCR instrument and incubate at 37°C for 3 to 6 hours.

[0077] 13-4, add 2 μl of Turbo DNase (from MEGAscript T7 kit, Ambion, cat. no. AM1334) to each sample.

[0078] 13-5, mix gently and incubate at 37°C for 15 min.

[0079] 13-6. Purify the DNase-treated reaction using the MEGAclear kit (Ambion, cat. no. AM1908); elute the modified mRNA with a total of 100 μl of elution buffer (elute twice with 50 μl of elution buffer).

[0080] 13-7, the purified mRNA nucleic acid molecules of each transcription factor were treated with phosphatase (Antarctic phosphatase (New England Biolabs, cat. no. M0289S).

[0081] 13-8. To each sample (~100 μl), add 11 μl of 10× phosphatase buffer, then add 2 μl of phosphatase; mix the samples gently and incubate at 37°C for 0.5-1 h.

[0082] 13-9, after elution, measure the concentration of modified mRNA in a NanoDrop spectrophotometer. The expected total yield should be ~50 ug (30-70 ug range; 100 ul elution volume for a 40 ul IVT reaction is 300-700 ng ul).

[0083] 13-10, the molar ratio of the mRNA nucleic acid molecule encoding the Oct4 transcription factor, the mRNA nucleic acid molecule encoding the Sox2 transcription factor, the mRNA nucleic acid molecule encoding the Klf4 transcription factor, the mRNA nucleic acid molecule encoding the Glis1 transcription factor, and the mRNA nucleic acid molecule encoding the Lin28 transcription factor is 3:1:1:1:1, and a skin reprogramming factor preparation is formed by adding citric acid-saline (10 mmol / L citrate, 130 mmol / L sodium chloride, nuclease-free distilled water, PH7.5), and the concentration is adjusted to 100 ng / μl.

[0084] Comparative Example 1: mRNA preparation encoding firefly fluorescent protein

[0085] An mRNA nucleic acid molecule encoding firefly fluorescent protein was prepared according to the method of Example 1. The firefly fluorescent protein full DNA template included a T7 promoter as shown in SEQ ID NO.10, a 5'UTR DNA sequence as shown in SEQ ID NO.1, a firefly fluorescent protein DNA sequence as shown in SEQ ID NO.8, a 3'UTR DNA sequence as shown in SEQ ID NO.2, and a poly-(a) tail. The firefly fluorescent protein full DNA template was then subjected to in vitro transcription to obtain an mRNA nucleic acid molecule encoding firefly fluorescent protein.

[0086] The mRNA nucleic acid molecule encoding the firefly fluorescent protein is dissolved in citric acid-physiological saline (10 mmol / L citrate, 130 mmol / L sodium chloride, nuclease-free distilled water, pH 7.5) to form an mRNA preparation encoding the firefly fluorescent protein.

[0087] Comparative Example 2: Blank Control Preparation

[0088] Citric acid-normal saline (10 mmol / L citrate, 130 mmol / L sodium chloride, nuclease-free distilled water, pH 7.5) was used as a blank control preparation.

[0089] Comparative Example 3: mRNA preparation encoding skin epidermal factor EGF

[0090] An mRNA nucleic acid molecule encoding the skin epidermal factor EGF was prepared according to the method of Example 1. The skin epidermal factor EGF full DNA template included a T7 promoter as shown in SEQ ID NO.10, a 5'UTR DNA sequence as shown in SEQ ID NO.1, a skin epidermal factor EGF DNA sequence as shown in SEQ ID NO.9, a 3'UTR DNA sequence as shown in SEQ ID NO.2, and a poly-(a) tail. The skin epidermal factor EGF full DNA template was then subjected to in vitro transcription to obtain an mRNA nucleic acid molecule encoding the skin epidermal factor EGF.

[0091] The mRNA nucleic acid molecule encoding the skin epidermal factor EGF is dissolved in citric acid-physiological saline (10 mmol / L citrate, 130 mmol / L sodium chloride, nuclease-free distilled water, pH 7.5) to form an mRNA preparation encoding the skin epidermal factor EGF.

[0092] Example 2: Human keratinocyte scratch test

[0093] This example uses HaCaT (human immortalized keratinocytes) as the experimental subject, and includes the following steps:

[0094] Step 1, cell culture and pretreatment: HaCaT cells were cultured in an appropriate culture medium and treated with 100 ng / ml of B18R recombinant protein overnight before transfection.

[0095] Step 2: Prepare the transfection RNA iMax (purchased from Life Technology) complex:

[0096] Dilute RNAiMax transfection reagent to an appropriate concentration in serum-free culture medium (such as Opti-MEM) according to the manufacturer's instructions.

[0097] The skin reprogramming factor preparation of Example 1 was mixed with RNAiMax transfection reagent to form a reprogramming mRNA transfection complex. The reprogramming mRNA transfection complex was incubated at room temperature for a period of time, generally 5-10 minutes.

[0098] The blank control preparation of Comparative Example 2 was mixed with RNAiMax transfection reagent to form a blank mRNA transfection complex. The blank mRNA transfection complex was incubated at room temperature for a period of time, generally 5-10 minutes.

[0099] The mRNA preparation encoding the epidermal growth factor (EGF) of Comparative Example 3 was mixed with RNAiMax transfection reagent to form an EGF mRNA transfection complex. The EGF mRNA transfection complex was incubated at room temperature for a period of time, generally 5-10 minutes.

[0100] Step 3, Transfection: Add the reprogramming mRNA transfection complex, blank mRNA transfection complex, and epidermal growth factor mRNA transfection complex to the cell culture medium, allowing each complex to contact the HaCaT cells. The three transfection complexes can be added dropwise to the culture dish, ensuring that the transfection reagent and mRNA are evenly distributed throughout the dish. Scrape the coating.

[0101] The results are shown in Figures 1 and 2.

[0102] Example 3: Human fibroblast scratch assay

[0103] This example uses human fibroblasts as experimental subjects and includes the following steps:

[0104] Step 1, cell culture and pretreatment: HaCaT cells were cultured in an appropriate culture medium and treated with 100 ng / ml of B18R recombinant protein overnight before transfection.

[0105] Step 2: Prepare the transfection RNA iMax (purchased from Life Technology) complex:

[0106] Dilute RNAiMax transfection reagent to an appropriate concentration in serum-free culture medium (such as Opti-MEM) according to the manufacturer's instructions.

[0107] The skin reprogramming factor preparation of Example 1 (total mRNA concentration of 10 ng / mL) was mixed with RNAiMax transfection reagent to form a low-concentration reprogramming mRNA transfection complex. The reprogramming mRNA transfection complex was incubated at room temperature for a period of time, generally 5-10 minutes.

[0108] The skin reprogramming factor preparation of Example 1 (total mRNA concentration of 20 ng / mL) was mixed with RNAiMax transfection reagent to form a high-concentration reprogramming mRNA transfection complex. The reprogramming mRNA transfection complex was incubated at room temperature for a period of time, generally 5-10 minutes.

[0109] The blank control preparation of Comparative Example 2 was mixed with RNAiMax transfection reagent to form a blank mRNA transfection complex. The blank mRNA transfection complex was incubated at room temperature for a period of time, generally 5-10 minutes.

[0110] The mRNA preparation encoding firefly fluorescent protein of Comparative Example 1 was mixed with RNAiMax transfection reagent to form a fluorescent protein mRNA transfection complex. The fluorescent protein mRNA transfection complex was incubated at room temperature for a period of time, generally 5-10 minutes.

[0111] Step 3, Transfection: Add low-concentration reprogramming mRNA transfection complex, high-concentration reprogramming mRNA transfection complex, blank mRNA transfection complex, and fluorescent protein mRNA transfection complex to the cell culture medium, allowing the low-concentration reprogramming mRNA transfection complex, high-concentration reprogramming mRNA transfection complex, blank mRNA transfection complex, and fluorescent protein mRNA transfection complex to contact the fibroblasts. The three transfection complexes can be added by directly adding them dropwise to the culture dish, ensuring that the transfection reagent and mRNA are evenly distributed in the culture dish, and scratching the coating layer.

[0112] The results are shown in Figures 3 and 4.

[0113] Example 4: Fluorescence expression experiment in mouse skin defect model

[0114] Establishment of Mouse Skin Defect Model: Select appropriate BALB / c mice to ensure they are healthy and meet experimental requirements. Before performing experimental procedures, anesthetize the mice using isoflurane anesthesia. Disinfect the mouse skin with an appropriate disinfectant (such as alcohol). Use a scalpel or scissors to create a skin defect on the back of the mouse.

[0115] The mRNA formulation encoding firefly fluorescent protein (Luci) from Comparative Example 1 (injection dose: 50 ng) and the blank control formulation from Comparative Example 2 were intradermally injected into mice bearing a skin defect model, and fluorescent protein expression was observed at different times post-injection. Expression of the mRNA formulation encoding Luci was quantitatively measured in living animals using a small animal imaging instrument, as shown in Figure 5.

[0116] Example 5: Wound healing experiment in mouse skin defect model

[0117] Establishment of Mouse Skin Defect Model: Select appropriate BALB / c mice to ensure they are healthy and meet experimental requirements. Before performing experimental procedures, anesthetize the mice using isoflurane anesthesia. Disinfect the mouse skin with an appropriate disinfectant (such as alcohol). Use a scalpel or scissors to create a skin defect on the back of the mouse.

[0118] The skin reprogramming factor preparation of Example 1 (mRNA injection amount 200 ng), the mRNA preparation encoding firefly fluorescent protein of Comparative Example 1 (mRNA injection amount 200 ng), the blank control preparation of Comparative Example 2, and the mRNA preparation encoding skin epidermal factor EGF of Comparative Example 3 (mRNA injection amount 200 ng) were intradermally injected into mice with skin defect models once every 3 days to treat the skin defects of the mice. The healing time and scar quality were evaluated and quantified using the internationally accepted Patient and Observer Scar Assessment Scale (POSAS) scoring system. The scar color, scar surface smoothness, scar thickness, pain, etc. were quantitatively evaluated. The results are shown in Figures 6, 7, and 8.

[0119] Example 6: Skin wound healing experiment using skin reprogramming factor preparations in different ratios

[0120] Establishment of Mouse Skin Defect Model: Select appropriate BALB / c mice to ensure they are healthy and meet experimental requirements. Before performing the experiment, anesthetize the mice using isoflurane anesthesia. Disinfect the mouse skin with an appropriate disinfectant (e.g., alcohol). Use a scalpel or scissors to create a skin defect on the back of the mouse.

[0121] This embodiment is divided into the following six experimental groups:

[0122] Experimental group 1 is a blank control preparation: the blank control preparation of comparative example 2 is used;

[0123] Experimental Group 2 was a reporter gene chemically modified mRNA: the mRNA preparation encoding firefly fluorescent protein of Comparative Example 1 was used;

[0124] Experimental group 3: OSKGL (3:1:1:1:1) chemically modified mRNA: the formulation of Example 1 was used;

[0125] Experimental Group 4 is an EGF mRNA preparation: the mRNA preparation encoding the skin epidermal factor EGF of Comparative Example 3 is used;

[0126] Experimental group 5 was OSKGL (0.5:1:1:1:1) chemically modified mRNA: the molar ratio of the mRNA nucleic acid molecule encoding the Oct4 transcription factor, the mRNA nucleic acid molecule encoding the Sox2 transcription factor, the mRNA nucleic acid molecule encoding the Klf4 transcription factor, the mRNA nucleic acid molecule encoding the Glis1 transcription factor, and the mRNA nucleic acid molecule encoding the Lin28 transcription factor was 0.5:1:1:1:1. By adding citric acid-normal saline (10 mmol / L citrate, 130 mmol / L sodium chloride, nuclease-free distilled water, pH 7.5), the skin reprogramming factor preparation was formed and the concentration was adjusted to 100 ng / μl.

[0127] Experimental group 6 was OSKGL (1:1:1:1:1) chemically modified mRNA: the molar ratio of the mRNA nucleic acid molecule encoding the Oct4 transcription factor, the mRNA nucleic acid molecule encoding the Sox2 transcription factor, the mRNA nucleic acid molecule encoding the Klf4 transcription factor, the mRNA nucleic acid molecule encoding the Glis1 transcription factor, and the mRNA nucleic acid molecule encoding the Lin28 transcription factor was 1:1:1:1:1. By adding citric acid-normal saline (10 mmol / L citrate, 130 mmol / L sodium chloride, nuclease-free distilled water, pH 7.5), the skin reprogramming factor preparation was formed and the concentration was adjusted to 100 ng / μl.

[0128] Mice with skin defect models were intradermally injected with 200 ng of mRNA in experimental groups 1, 2, 3, 4, 5, and 6, respectively (the mRNA injection amount in each experimental group was 200 ng), once every 3 days to treat the skin defects of the mice. Specimens were collected at the corresponding time points during the healing period and sent to relevant biological companies such as My DNAage (https: / / www.mydnage.com_) for Horvath epigenetic clock calculation. The results are shown in Figure 9.

[0129] Experimental results and analysis:

[0130] Figure 1 is an inverted microscope image of cell proliferation and migration in the human keratinocyte scratch experiment in Example 2. As shown in Figure 1, transfection of reprogramming factor mRNA promotes HaCaT proliferation and migration, and HaCaT transfected with reprogramming factor mRNA has the strongest migration ability. The scratch of HaCaT transfected with reprogramming factor mRNA healed 24 hours after transfection, while the scratch of HaCaT transfected with skin epidermal factor EGF mRNA and HaCaT transfected with a blank control preparation did not heal, indicating that transfection of reprogramming factor mRNA can promote HaCaT migration more than transfection of skin epidermal factor EGF mRNA, and further indicating that transfection of reprogramming factor mRNA has a better effect of promoting wound healing. Among them, the reprogramming factor mRNA includes mRNA nucleic acid molecules encoding Oct4 transcription factor, mRNA nucleic acid molecules encoding Sox2 transcription factor, mRNA nucleic acid molecules encoding Klf4 transcription factor, mRNA nucleic acid molecules encoding Glis1 transcription factor, and mRNA nucleic acid molecules encoding Lin28 transcription factor.

[0131] Figure 2 is a quantitative bar graph of the cell proliferation and migration results of the human keratinocyte scratch test in Example 2. As shown in Figure 2, the scratch healing rates of HaCaT cells transfected with reprogramming factor mRNA were the highest 12 hours, 16 hours, and 24 hours after transfection, respectively. This indicates that transfection of reprogramming factor mRNA can promote the migration of HaCaT cells more than transfection of epidermal growth factor (EGF) mRNA, and further indicates that transfection of reprogramming factor mRNA has a better effect in promoting wound healing.

[0132] Figure 3 is an inverted microscope image of cell proliferation and migration of the human fibroblast scratch experiment in Example 3. The transfection of high-concentration reprogramming factor mRNA and low-concentration reprogramming factor mRNA can promote the proliferation and migration of human fibroblasts, respectively. Moreover, the human fibroblasts transfected with high-concentration reprogramming factor mRNA and the human fibroblasts transfected with low-concentration reprogramming factor mRNA have the strongest migration ability. The scratch healing effects of human fibroblasts transfected with high-concentration reprogramming factor mRNA and the human fibroblasts transfected with low-concentration reprogramming factor mRNA are higher than the scratch healing effects of human fibroblasts transfected with fluorescent protein mRNA and the human fibroblasts transfected with blank control preparation, respectively.

[0133] Figure 4 is a quantitative bar graph of the cell proliferation and migration results of the human fibroblast scratch experiment in Example 3. The scratch healing rates of human fibroblasts transfected with high-concentration reprogramming factor mRNA and human fibroblasts transfected with low-concentration reprogramming factor mRNA were higher 12 hours, 16 hours, 24 hours, and 48 hours after transfection, respectively, indicating that transfection of reprogramming factor mRNA can promote the migration of human fibroblasts better than transfection of fluorescent protein mRNA, and further indicating that transfection of reprogramming factor mRNA has a better effect in promoting wound healing.

[0134] Figure 5 is the distribution diagram of fluorescent protein expression of Example 4. As shown in Figure 5, the mRNA preparation encoding firefly fluorescent protein is efficiently and persistently expressed locally in the skin wound. Expression begins within 3 hours after injection and lasts for at least 5 days. It is confined to the skin wound and there is no systemic expression. This confirms that the mRNA preparation of the present application can be controllably and highly targeted delivered to the skin wound, and the mRNA preparation is successfully established.

[0135] Specifically, in a mouse skin lesion model, no firefly luciferin expression was observed after intradermal injection of a blank control formulation. In a mouse skin lesion model, no firefly luciferin expression was observed after intradermal injection of an mRNA formulation encoding firefly fluorescent protein. Firefly fluorescent protein mRNA was efficiently expressed locally in the skin lesions, with no expression elsewhere. Fluorescent protein expression began within 3 hours, reaching and maintaining a peak over several days. After 5 days, firefly fluorescent protein mRNA was metabolized along a normal metabolic pathway, indicating that the mRNA formulation disclosed herein has a fixed half-life and is safe and controllable.

[0136] Please refer to Figures 6 to 8 . In the mouse skin injury model, reprogramming factor mRNA can promote rapid healing of skin wounds and reduce scars. Specifically, in the mouse skin injury model, reprogramming factor mRNA promotes the healing of skin injury wounds more quickly than firefly fluorescent protein mRNA and epidermal growth factor (EGF) mRNA.

[0137] In Example 5, the efficacy results of skin epidermal factor EGF mRNA, reprogramming factor mRNA, firefly fluorescent protein mRNA and blank control preparation in the mouse skin injury model are as follows:

[0138] Please refer to Figure 6, which shows changes in the wound surface after treatment with reprogramming factor mRNA, firefly fluorescent protein mRNA, and epidermal growth factor (EGF) mRNA in a mouse skin injury treatment model. The results suggest that reprogramming factor mRNA promotes wound healing more rapidly than firefly fluorescent protein mRNA and epidermal growth factor (EGF) mRNA. Furthermore, the quality of wound healing, i.e., scar formation, at each time point is also superior to that of firefly fluorescent protein mRNA and epidermal growth factor (EGF) mRNA.

[0139] Please refer to Figure 7, which shows the quantitative results of wound healing in a mouse skin injury treatment model treated with reprogramming factor mRNA, firefly fluorescent protein mRNA, and epidermal growth factor (EGF) mRNA. The results indicate that compared with firefly fluorescent protein mRNA, epidermal growth factor (EGF) mRNA can accelerate the healing of mouse skin wounds and improve wound quality; and reprogramming factor mRNA has a more excellent therapeutic effect than epidermal growth factor (EGF) mRNA, and can further accelerate wound healing and further improve the quality of wound healing than epidermal growth factor (EGF) mRNA.

[0140] Please refer to Figure 8, which shows the quantitative results of wound healing quality in a mouse skin injury treatment model treated with reprogramming factor mRNA, firefly fluorescent protein mRNA, and epidermal growth factor (EGF) mRNA. The wound healing quality was evaluated using the internationally accepted wound assessment system POSAS. The evaluation results indicate that compared with firefly fluorescent protein mRNA, epidermal growth factor (EGF) mRNA can accelerate the healing of mouse skin wounds and improve wound quality; and reprogramming factor mRNA has a more excellent therapeutic effect than epidermal growth factor (EGF) mRNA, and can further accelerate wound healing and improve the quality of wound healing compared with epidermal growth factor (EGF) mRNA.

[0141] Please refer to Figure 9, which shows the quantitative results of the Horvath apparent clock of the mouse skin injury treatment model, the reprogramming factor mRNA of Example 1, the firefly fluorescent protein mRNA of Comparative Example 2, and the epidermal growth factor EGF mRNA of Comparative Example 3 for treating wounds. The evaluation results suggest that the programming factor mRNA (3:1:1:1:1) treatment group of Example 1 can reduce the Horvath apparent clock age of mouse skin wounds and shorten the wound healing time compared with the firefly fluorescent protein mRNA of Comparative Example 2, the epidermal growth factor EGF mRNA of Comparative Example 3, the programming factor mRNA (1:1:1:1:1), and the programming factor mRNA (0.5:1:1:1:1:1).

[0142] The above is only an implementation method of the present application. It should be pointed out that for ordinary technicians in this field, improvements can be made without departing from the creative concept of the present application, but these all fall within the scope of protection of the present application.

Claims

1. Use of a skin reprogramming factor preparation in the preparation of a drug for promoting skin wound healing, characterized in that: The skin reprogramming factor preparation includes 2 to 4 molar parts of mRNA nucleic acid molecules encoding Oct4 transcription factor, 1 molar part of mRNA nucleic acid molecules encoding Sox2 transcription factor, 1 molar part of mRNA nucleic acid molecules encoding Klf4 transcription factor, 1 molar part of mRNA nucleic acid molecules encoding Glis1 transcription factor and 1 molar part of mRNA nucleic acid molecules encoding Lin28 transcription factor.

2. Use of the skin reprogramming factor preparation according to claim 1 in the preparation of a drug for promoting skin wound healing, characterized in that: The skin reprogramming factor preparation includes 3 molar parts of mRNA nucleic acid molecules encoding Oct4 transcription factor, 1 molar part of mRNA nucleic acid molecules encoding Sox2 transcription factor, 1 molar part of mRNA nucleic acid molecules encoding Klf4 transcription factor, 1 molar part of mRNA nucleic acid molecules encoding Glis1 transcription factor and 1 molar part of mRNA nucleic acid molecules encoding Lin28 transcription factor.

3. Use of the skin reprogramming factor preparation according to claim 1 in the preparation of a drug for promoting skin wound healing, characterized in that: The skin reprogramming factor preparation further includes a first solvent, wherein the concentration of citrate in the first solvent is 8 to 12 mmol / L, and the concentration of sodium chloride in the first solvent is 120 to 140 mmol / L.

4. Use of the skin reprogramming factor preparation according to claim 3 in the preparation of a drug for promoting skin wound healing, characterized in that: The concentration of citrate in the first solvent is 10 mmol / L, the concentration of sodium chloride in the first solvent is 130 mmol / L, and the pH value of the first solvent is 7.

5.

5. Use of the skin reprogramming factor preparation according to claim 1 in the preparation of a drug for promoting skin wound healing, characterized in that: Each of the mRNA nucleic acid molecules comprises a 5' cap structure, a 5' UTR sequence, an mRNA coding sequence of a corresponding transcription factor, a 3' UTR sequence and a polyadenylation sequence.

6. Use of the skin reprogramming factor preparation according to claim 1 in the preparation of a drug for promoting skin wound healing, characterized in that: Part or all of the nucleotides in the mRNA nucleic acid molecule encoding the Oct4 transcription factor, the mRNA nucleic acid molecule encoding the Sox2 transcription factor, the mRNA nucleic acid molecule encoding the Klf4 transcription factor, the mRNA nucleic acid molecule encoding the Glis1 transcription factor, and the mRNA nucleic acid molecule encoding the Lin28 transcription factor are chemically modified to improve the stability of the mRNA nucleic acid molecules in vivo.

7. Use of the skin reprogramming factor preparation according to claim 6 in the preparation of a drug for promoting skin wound healing, characterized in that: The chemical modification includes replacing 100% of the cytosine in the mRNA nucleic acid molecules encoding the Oct4 transcription factor, the mRNA nucleic acid molecules encoding the Sox2 transcription factor, the mRNA nucleic acid molecules encoding the Klf4 transcription factor, the mRNA nucleic acid molecules encoding the Glis1 transcription factor, and the mRNA nucleic acid molecules encoding the Lin28 transcription factor with 5-methylcytosine, and replacing 100% of the uracil in the mRNA nucleic acid molecules encoding the Oct4 transcription factor, the mRNA nucleic acid molecules encoding the Sox2 transcription factor, the mRNA nucleic acid molecules encoding the Klf4 transcription factor, the mRNA nucleic acid molecules encoding the Glis1 transcription factor, and the mRNA nucleic acid molecules encoding the Lin28 transcription factor with N1-methylpseudouridine.

8. Use of the skin reprogramming factor preparation according to claim 6 in the preparation of a drug for promoting skin wound healing, characterized in that: The DNA sequence of the Oct4 transcription factor is shown in SEQ ID NO.3, the DNA sequence of the Sox2 transcription factor is shown in SEQ ID NO.4, the DNA sequence encoding the Klf4 transcription factor is shown in SEQ ID NO.5, the DNA sequence of the Glis1 transcription factor is shown in SEQ ID NO.6, and the DNA sequence of the Lin28 transcription factor is shown in SEQ ID NO.

7.

9. Use of the skin reprogramming factor preparation according to claim 8 in the preparation of a drug for promoting skin wound healing, characterized in that: The DNA sequence of the 5'UTR sequence is shown as SEQ ID NO.1, and the DNA sequence of the 3'UTR sequence is shown as SEQ ID NO.

2.

10. Use of the skin reprogramming factor preparation according to any one of claims 1 to 9 in the preparation of a drug for promoting skin wound healing, characterized in that: The skin reprogramming factor preparation is an injection.

Images