Skin reprogramming factor formulation, biological material, and uses

Abstract

Provided are a skin reprogramming factor formulation, a biological material, and uses. 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 an 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 an Lin28 transcription factor. When the skin reprogramming factor formulation is introduced into the skin, in situ reversal of skin aging is achieved, the expression of skin aging related genes is down-regulated, and a good skin wrinkle eliminating effect is achieved; in addition, hair follicle related genes are up-regulated, so that hair follicle proliferation can be promoted, and hair growth can be promoted.

Description

Skin reprogramming factor preparations, biomaterials and uses Technical Field

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

[0002] Skin aging is manifested as the gradual loss of elasticity and a decrease in collagen content, leading to sagging skin and the formation of wrinkles and fine lines, especially on the forehead, corners of the eyes and neck. At the same time, muscle contractions caused by repetitive facial expressions can also cause wrinkles.

[0003] Currently, botulinum toxin injections are the leading non-surgical wrinkle reduction method. While they effectively reduce expression wrinkles, they also have drawbacks. The effects are temporary, lasting an average of three to four months. Patients require regular injections to maintain a smooth, youthful appearance. Without regular injections, wrinkles will reappear. Repeated botulinum toxin injections can also induce an immune response, rendering them ineffective.

[0004] Hundreds of millions of men, women, and even children worldwide suffer from hair loss, which can be caused by a variety of factors, including genetics, aging, childbirth, cancer treatment, burns, and stress. While hair loss doesn't pose a serious health concern, it can permanently alter a person's appearance and can lead to emotional distress, trauma, and even depression.

[0005] At present, the methods for treating hair loss are very limited, mainly including drug treatment (minoxidil, finasteride), surgical treatment (hair transplantation), physical therapy (traditional Chinese medicine acupuncture, low-frequency laser therapy), etc., which cannot prevent hair loss in the long term, let alone achieve hair follicle regeneration.

[0006] Summary of the Invention

[0007] In view of the above problems, the embodiments of the present application provide a skin reprogramming factor preparation, biomaterial and use to solve the above technical problems.

[0008] In a first aspect, an 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.

[0009] 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.

[0010] 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.

[0011] 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.

[0012] 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.

[0013] 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.

[0014] 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.

[0015] In a second aspect, an embodiment of the present application provides a biomaterial, wherein the biomaterial is host skin cells comprising the above-mentioned skin reprogramming factor preparation.

[0016] In a third aspect, embodiments of the present application provide a use of the aforementioned skin reprogramming factor preparation or the aforementioned biomaterial in preparing a skin cell reprogramming reagent.

[0017] In a fourth aspect, an embodiment of the present application provides a use of the above-mentioned skin reprogramming factor preparation or the above-mentioned biomaterial in the preparation of skin anti-aging drugs or hair growth drugs.

[0018] The skin reprogramming factor preparation, biomaterial and use provided in the embodiments of the present application include 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 the skin, it reverses skin aging in situ, downregulates the expression of skin aging-related genes, and has a good effect in eliminating skin wrinkles. At the same time, it upregulates hair follicle-related genes, which can promote hair follicle proliferation and promote hair growth.

[0019] 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

[0020] FIG1 shows the results of the human in vitro skin fluorescence experiment in Example 2 of the present application.

[0021] FIG2 shows the results of the human ex vivo skin injection experiment in Example 3 of the present application.

[0022] FIG3 shows the results of the skin injection experiment on the skin defect model after skin transplantation in immunodeficient mice in Example 4 of the present application.

[0023] FIG4 shows the experimental results of skin injection of skin reprogramming factor preparations with different ratios in Example 5 of the present application. DETAILED DESCRIPTION

[0024] 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.

[0025] 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.

[0026] 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.

[0027] 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.

[0028] 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.

[0029] 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.

[0030] The experimental methods in the following examples are conventional methods unless otherwise specified.

[0031] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0032] 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.

[0033] In this embodiment, a specific skin reprogramming factor preparation is formed by adjusting the molar ratio of mRNA nucleic acid molecules expressing different transcription factors. After being introduced into the skin, it reverses skin aging in situ and downregulates the expression of genes related to skin aging, which has a better effect in eliminating skin wrinkles. At the same time, hair follicle-related genes are upregulated, which can promote hair follicle proliferation and hair growth.

[0034] 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.

[0035] 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.

[0036] In this embodiment, the first solvent can make each mRNA nucleic acid molecule less susceptible to degradation.

[0037] 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.

[0038] 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.

[0039] 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.

[0040] 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.

[0041] 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.

[0042] Example 1: Preparation of skin reprogramming factor preparation

[0043] The skin reprogramming factor preparation of this embodiment can be prepared according to the following steps:

[0044] S11, Preparation of DNA templates for each transcription factor

[0045] 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;

[0046] 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;

[0047] 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;

[0048] 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;

[0049] 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;

[0050] 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.

[0051] S12, preparation of DNA templates for each transcription factor

[0052] 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.

[0053] Prepare PCR premix according to Table 1 (total volume 200 μl, 25 μl for each of eight reactions)

[0054] Table 1. Composition of PCR master mix

[0055] Among them, the tailing primer-F1 is shown as SEQ ID NO.11, and the tailing primer-T120 is shown as SEQ ID NO.9.

[0056] Perform PCR using the conditions in Table 2:

[0057] Table 2. Tailing PCR conditions

[0058] Check the quality of PCR products by gel electrophoresis.

[0059] 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.

[0060] S13, in vitro transcription synthesis of each transcription factor mRNA nucleic acid molecule

[0061] 13-1. Assemble the mRNA cap structure and nucleotide mixture according to Table 3:

[0062] 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).

[0063] Table 3. mRNA cap structure and nucleotide mixture

[0064] 13-2. Assemble the mRNA in vitro transcription system according to Table 4:

[0065] Table 3. mRNA in vitro transcription system

[0066] 13-3. Place the reaction in a PCR instrument and incubate at 37°C for 3 to 6 hours.

[0067] 13-4, add 2 μl of Turbo DNase (from MEGAscript T7 kit, Ambion, cat. no. AM1334) to each sample.

[0068] 13-5, mix gently and incubate at 37°C for 15 min.

[0069] 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).

[0070] 13-7, the purified mRNA nucleic acid molecules of each transcription factor were treated with phosphatase (Antarctic phosphatase (New England Biolabs, cat. no. M0289S).

[0071] 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.

[0072] 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).

[0073] 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-normal saline (10 mmol / L citrate, 130 mmol / L sodium chloride, nuclease-free distilled water, pH 7.5), and the concentration is adjusted to 100 ng / μl.

[0074] Example 2: Verification of intradermal injection of mRNA formulation encoding firefly fluorescent protein

[0075] 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.

[0076] 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.

[0077] The mRNA preparation encoding firefly fluorescent protein was injected into human ex vivo skin, and the expression of the mRNA preparation encoding firefly fluorescent protein was quantitatively detected using an IVIS imager. The specific steps were as follows: the volunteer's full-thickness skin was surgically incised and transported to the laboratory. After rinsing with saline, the fat tissue was removed with dissecting scissors, retaining the epidermis and dermis. The cells were cultured in 10% fetal bovine serum DEME medium and a 5% CO2 cell culture incubator to form a human ex vivo skin culture model. The mRNA preparation encoding firefly fluorescent protein was injected intradermally at the junction of the epidermis and dermis. After 6 hours, the firefly fluorescent protein substrate was added, and the fluorescence intensity of the firefly fluorescent protein was detected using an IVIS in vitro live imager.

[0078] Example 3: Verification of Intradermal Injection of Skin Reprogramming Factor Preparation

[0079] The skin reprogramming factor preparation prepared in Example 1 was injected into ex vivo human skin (for the preparation of ex vivo human skin, see the description in Example 2) to reverse skin aging in situ. An empty mRNA preparation and the mRNA preparation encoding firefly fluorescent protein of Example 2 were used as controls. After collecting skin tissue, quantitative PCR was performed to measure the aging marker P16, the skin epigenetic marker H3K9me3, the skin cell proliferation marker K67, skin collagens I and VI, the extracellular matrix MMP1 and MMP9, and the oxygen free radical SOD3. The specific steps are as follows:

[0080] After the specimens were collected, total mRNA was extracted using Trizol and expressed according to In Vitrogen SuperScript TM Perform one-step reverse transcription-PCR according to the IV One-Step RT-PCR System instruction manual. Configure the one-step reverse transcription-PCR reaction system and use the SybrGreen method. Perform the reaction on an ABI QuantStudio 5 quantitative PCR instrument. Process the data using double delta and output quantitative gene expression data.

[0081] Example 4: Establishment of a skin defect model after ex vivo human skin-immunodeficient mouse skin transplantation and skin injection of skin reprogramming factor preparations

[0082] SCUB3 mRNA preparation and reporter gene mRNA preparation were used as comparisons. The SCUB3 mRNA preparation and reporter gene mRNA preparation were prepared according to the method of Example 1.

[0083] The SCUB3-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 DNA sequence encoding SCUB3, a 3' UTR DNA sequence as shown in SEQ ID NO. 2, and a poly-(a) tail. The SCUB3-DNA template is then subjected to in vitro transcription to produce an mRNA nucleic acid molecule encoding SCUB3. The mRNA nucleic acid molecule encoding SCUB3 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 a SCUB3 mRNA preparation.

[0084] 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. The mRNA nucleic acid molecule encoding firefly fluorescent protein was 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 firefly fluorescent protein.

[0085] The skin reprogramming factor preparation, SCUB3 mRNA preparation, and Luc mRNA preparation (the mRNA preparation encoding firefly fluorescent protein of Example 2) were injected into the skin to reverse skin aging in situ and promote human skin repair. The effects were then evaluated. The specific steps were as follows:

[0086] First, a skin defect model was established after human ex vivo skin-immunodeficient mouse skin transplantation: NOD-Prkdcscid / Smoc immunodeficient mice were purchased from the corresponding biological company, and the health status and immunodeficiency characteristics of the mice were determined before the experiment began.

[0087] Donor skin extraction: The skin tissue removed from a volunteer during surgery is used as the donor skin. Strict aseptic operating procedures are followed during the operation to reduce the risk of infection.

[0088] Donor skin trimming: Remove fat and other tissue and trim the extracted skin tissue into a 2x2 cm piece to fit the mouse's skin defect area. Processing the skin piece may include washing, hair removal, and fat tissue removal to ensure the purity of the graft.

[0089] Mouse preparation: Anesthetize immunodeficient mice and perform local disinfection on the target skin defect area. Ensure that the mice are pain-free and comfortable.

[0090] Skin transplantation: Carefully place the processed skin piece onto the skin defect area of ​​the mouse. Secure the skin piece to the defect area using sutures or biological glue. Create an approximately 1 mm lesion on ex vivo human skin and cover with a sterile dressing. Observe regularly and collect samples at appropriate time points.

[0091] Second, HE staining:

[0092] First, it is necessary to obtain tissue sections that have been fixed and embedded in 4% PFA and then undergo a dehydration and embedding process to finally obtain sections; dewaxing and dehydration: immerse the sections of the wax block in the wax remover Xylene to remove the wax.

[0093] Water washing: Wash the dewaxed sections under running tap water to completely remove the dewax remover residue.

[0094] Staining: The sections are immersed in a series of stains, starting with hematoxylin, which stains the nucleus and surrounding material. The sections are then transferred to eosin solution, which stains the cytoplasm and extracellular matrix.

[0095] Dehydration: Immerse the stained sections in different concentrations of alcohol (such as 70% alcohol, 95% alcohol and absolute alcohol) in turn to dehydrate the sections.

[0096] Detergent treatment: Immerse the sections in an appropriate detergent, such as Xylene or other detergents, to remove the alcohol and make the sections transparent.

[0097] Mounting: Place the sections in an appropriate medium, such as a clear mounting medium (e.g., Mounting Medium) between a glass slide and a cover slip, then cover with a coverslip.

[0098] After completing the above steps, the sections are ready. Using a microscope, HE-stained sections can be used to observe the staining of cell nuclei (blue or purple), cytoplasm and extracellular matrix (pink), and details of skin tissue structure such as anagen hair follicles. Image J software is used to quantify epidermal thickness and anagen hair follicles, and Graphpad Prism software is used for statistics and data output.

[0099] Third, Masson staining:

[0100] First, tissue sections are obtained that have been fixed and embedded in 4% PFA, followed by dehydration and embedding. Finally, sections are dewaxed and dehydrated: sections from the wax block are immersed in Xylene dewaxing agent to remove the wax. Washing: dewaxed sections are washed under running tap water to completely remove any residual dewaxing agent.

[0101] Acid staining: Immerse the sections in an acidic stain (e.g., acid carmine) to stain the cytoplasm and nuclei. The soaking time in the stain should be determined according to standard laboratory procedures. Washing: Wash the stained sections under running tap water to remove excess stain.

[0102] Cobalt acid staining: Immerse sections in a stain containing cobalt acid to stain collagen fibers. Cobalt acid stain is a blue dye that gives collagen fibers a blue hue. Washing: Wash the stained sections under running tap water to remove excess stain. Acid wash: Immerse sections in a dilute acidic detergent (such as acetic acid) to remove any cobalt acid residue.

[0103] Blue staining: Immerse the sections in a stain containing hematoxylin and hematoxylin. Hematoxylin is a blue stain used to stain cell nuclei. Washing: Wash the stained sections under running tap water to remove excess stain. Dehydration: Dehydrate the sections by immersing them in different concentrations of alcohol (e.g., 70% alcohol, 95% alcohol, and absolute alcohol). Detergent treatment: Immerse the sections in an appropriate detergent, such as Xylene or other detergents, to remove the alcohol and render the sections transparent.

[0104] Mounting: Place the sections in an appropriate medium, such as a clear mounting medium (e.g., Mounting Medium) between a glass slide and a cover slip, then cover with a coverslip.

[0105] After completing the above steps, the sections are ready for observation. Using a microscope, the Masson-stained sections can be used to observe the staining of collagen fibers (blue), the staining of cell nuclei (purple), and the details of tissue structure, and quantified using Image J software.

[0106] Immunohistochemistry: (a) Obtaining tissue sections: First, tissue sections that have been fixed and embedded in 4% PFA undergo a dehydration and embedding process to obtain sections. (b) Wax block treatment: Wax block sections are immersed in a wax remover to remove the wax with Xylene. (c) Antigen retrieval: Antigen activity in the sections is restored using heat treatment. (d) Protein blocking: To reduce nonspecific binding, nonspecific binding sites in the tissue sections are blocked using bovine serum albumin (BSA) and coated onto the sections. (e) Antibody incubation: A solution of the target antibody, anti-Oct4 antibody (R&D Systems), is added dropwise to the sections and incubated overnight at 4°C to allow the antibody to bind specifically to the Oct4 protein in the sections. (f) Washing: Following the antibody incubation, multiple washes are performed using a buffer solution such as PBS to remove unbound antibody and other nonspecific binding products. (g) Secondary antibody incubation: Sections are incubated with the corresponding specific secondary antibody (R&D Systems) at 4°C overnight. (h) Washing: After incubation with the secondary antibody, multiple washes are performed using a buffer solution or PBS to remove unbound secondary antibody and other non-specific binders. (i) Visualization staining: Depending on the type of secondary antibody, an enzyme-labeled horseradish peroxidase is used, and the enzyme substrate is added to the section to produce a visible staining reaction. (j) Washing and mounting: Finally, a final washing step is performed to remove excess staining material and substrate. The section is then placed in an appropriate medium, such as a transparent mounting medium (such as Mounting Medium) between a glass slide and a cover slide, and then covered with a cover slide. (k) After completing the above steps, the section is ready to be examined using a microscope to confirm the distribution and localization of Oct4, as well as other specific structures that specifically bind to the antibody.

[0107] Example 5: Skin injection experiment of skin reprogramming factor preparations with different ratios

[0108] This example is divided into the following four experimental groups:

[0109] Experimental group 1: reporter gene chemically modified mRNA: the preparation of Example 2 was used;

[0110] Experimental group 2: OSKGL (3:1:1:1:1) chemically modified mRNA: the formulation of Example 1 was used;

[0111] Experimental group 3 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.

[0112] Experimental group 4 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.

[0113] Experimental Group 1, Experimental Group 2, Experimental Group 3, and Experimental Group 4 were injected intradermally to reverse skin aging in situ, promote human skin repair, and evaluate the effects. The specific steps are as follows:

[0114] First, a skin defect model was established after human ex vivo skin-immunodeficient mouse skin transplantation: NOD-Prkdcscid / Smoc immunodeficient mice were purchased from the corresponding biological company, and the health status and immunodeficiency characteristics of the mice were determined before the experiment began.

[0115] Donor skin extraction: The skin tissue removed from a volunteer during surgery is used as the donor skin. Strict aseptic operating procedures are followed during the operation to reduce the risk of infection.

[0116] Donor skin trimming: Remove fat and other tissue and trim the extracted skin tissue into a 2x2 cm piece to fit the mouse's skin defect area. Processing the skin piece may include washing, hair removal, and fat tissue removal to ensure the purity of the graft.

[0117] Mouse preparation: Anesthetize immunodeficient mice and perform local disinfection on the target skin defect area. Ensure that the mice are pain-free and comfortable.

[0118] Skin grafting: Carefully place the processed skin piece onto the skin defect area of ​​the mouse. Secure the skin piece to the defect area using sutures or biological glue, cover with a sterile dressing, and observe regularly and take samples at appropriate time points.

[0119] Injection of different mRNA preparations: Four different treatments were injected intradermally into the above-mentioned transplanted human skin, namely experimental group 1, experimental group 2, experimental group 3 and experimental group 4. Samples were collected 10 days after injection and quantitative PCR was performed for the aging indicator P16 gene, epigenetic indicator H3K9me3 gene, wrinkle indicator collagen VI gene, wrinkle metalloproteinase gene and oxygen free radical indicator SOD2 gene.

[0120] Example 2 Experimental results:

[0121] Please refer to Figure 1 , where Part A of Figure 1 is a schematic diagram of ex vivo skin culture, and Part B of Figure 1 is a schematic diagram of ex vivo skin culture; Part C of Figure 1 shows that firefly luciferin mRNA preparation can be efficiently expressed 6 hours after being injected into the skin.

[0122] Example 3 experimental results:

[0123] As shown in Figure 2, the expression of various indicators of the empty mRNA preparation (NC), the mRNA preparation encoding firefly fluorescent protein (Luc mRNA), and the skin reprogramming factor preparation (OSKGL mRNA1 and OSKGL mRNA2 are the preparations of Example 1) after 5 days of partial reprogramming of human skin showed that the expression of the skin tissue aging indicator P16 gene was downregulated (A), the epigenetic rejuvenation indicator H3K9me3 gene expression was downregulated (B), the cell proliferation indicator K67 expression was upregulated (C), collagen I and collagen VI were upregulated (D), the extracellular matrix MMP1, MMP2 gene expression was downregulated (E), and the oxygen free radical indicator SOD2 gene expression was downregulated (F).

[0124] Example 4 Experimental results:

[0125] As shown in Figure 3, A: Immunohistochemistry in a human skin transplant model showed that the reprogramming factor OSKGL mRNA formulation of Example 1 was highly expressed throughout the skin layer 6 hours after injection, with expression localized to the skin. B: In a human skin transplant model, typical results of HE and Masson fiber staining showed that injection of the reprogramming factor OSKGL mRNA formulation of Example 1 could drive partial reprogramming throughout the skin layer and increase epidermal thickness. C: Quantitative analysis using Image J software showed that compared to a blank mRNA formulation, a firefly luciferin mRNA formulation, and a SCUBE3 mRNA formulation, the reprogramming factor OSKGL mRNA of Example 1 increased epidermal thickness and eliminated wrinkles. D: Quantitative analysis using pathological sections and Image J software showed that compared to a blank mRNA formulation, a firefly luciferin mRNA formulation, and a SCUBE3 mRNA formulation, the reprogramming factor OSKGL mRNA formulation of Example 1 significantly increased the number of anagen hair follicles and promoted hair growth. Compared to the SCUBE3 mRNA formulation, the reprogramming factor OSKGL mRNA formulation of Example 1 significantly increased the number of anagen hair follicles and promoted hair growth.

[0126] Example 5 Experimental results:

[0127] As shown in Figure 4, the mRNA preparation with a specific molar ratio of 3:1:1:1:1 of the reprogramming OSKGL factor was compared with the reporter gene mRNA preparation, the mRNA preparation with a specific molar ratio of 1:1:1:1:1 of the reprogramming OSKGL factor was compared with the mRNA preparation with a specific molar ratio of 0.5:1:1:1:1:1 of the reprogramming OSKGL factor. The mRNA preparation can significantly reduce the aging indicator P16 gene, significantly reduce the extracellular matrix metalloproteinase gene, significantly reduce the oxygen free radical indicator SOD2 gene, significantly increase the epigenetic youth indicator H3K9me3 gene expression, and significantly increase the skin collagen indicator collagen VI gene expression.

[0128] The skin reprogramming factor preparation prepared in the examples of the present application can effectively affect the entire skin gene expression pattern, activate the skin stem cell-related gene network, achieve the resetting of the cell function of the entire skin layer, reverse skin aging in situ, restore the skin to a youthful state, reorganize the cell function of the entire skin layer, increase skin collagen fibers, reduce the content of oxygen free radicals, rejuvenate epigenetic markers, and downregulate aging-related genes. The overall effect can provide a safer and more effective method for eliminating wrinkles.

[0129] The skin reprogramming factor preparation prepared in the embodiments of the present application can reverse skin aging in situ in the skin, upregulate hair follicle-related genes, and significantly increase the number of hair follicles in the growth phase, which can promote hair growth and / or improve the efficacy of hair transplantation surgery and / or unexpectedly increase the effect of chemically modified mRNA of SCUBE3 protein (hair follicle growth-promoting gene) to increase the number of hair follicles in the growth phase and promote hair growth.

[0130] 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. A skin reprogramming factor preparation, characterized in that: It 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. The skin reprogramming factor preparation according to claim 1, characterized in that It 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. The skin reprogramming factor preparation according to claim 1, 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. The skin reprogramming factor preparation according to claim 3, 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. The skin reprogramming factor preparation according to claim 1, 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. The skin reprogramming factor preparation according to claim 1, 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. The skin reprogramming factor preparation according to claim 6, 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. A biomaterial, characterized in that The biological material is host skin cells comprising the skin reprogramming factor preparation according to any one of claims 1 to 7.

9. Use of the skin reprogramming factor preparation according to any one of claims 1 to 7 or the biomaterial according to claim 8 in preparing a skin cell reprogramming reagent.

10. Use of the skin reprogramming factor preparation according to any one of claims 1 to 7 or the biomaterial according to claim 8 in the preparation of skin anti-aging drugs or hair growth drugs.

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