Type III collagen mRNA (messenger Ribonucleic Acid) medicine and application thereof

Abstract

The invention discloses an mRNA (messenger Ribonucleic Acid) medicine of type III collagen and application of the mRNA medicine, and belongs to the technical field of biological medicines. The method comprises the following steps: carrying out mRNA (messenger ribonucleic acid) coding optimization design on the basis of an amino acid sequence of human type III collagen, and carrying out in-vitro expression screening to obtain a candidate molecule Conus001 with high expression performance. Wild-type mouse in-vivo experiments prove that the molecule can significantly improve the expression level of III-type collagen, and subsequent mouse pharmacodynamic studies show that the molecule has a definite ultraviolet damage protection function. Experimental data reveals that through gene delivery of Conus001, expression of III-type collagen is promoted, ultraviolet damage resistance of an organism can be effectively improved, and skin quality and skin elasticity can be recovered. A novel treatment scheme is provided for delaying senescence of human beings.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to a type III collagen mRNA drug and its application. Background Technology

[0002] Collagen molecules are composed of three amino acid chains and are one of the body's main structural proteins, accounting for about 30% of all proteins in the body. Collagen forms the framework of the dermis and basal layer, bones, internal organs, blood vessels, tendons, and more. The normal triple helix conformation of collagen is the basis of its physicochemical properties and biological activity, giving it characteristics such as high tensile strength and biodegradability. There are many types of collagen in the human body, which can be classified into more than 30 types, including Type I, Type II, Type III, Type IV, and Type V, based on their physiological functions and roles. Type I collagen accounts for about 80% of the total collagen in the skin, giving the skin tensile strength and resilience, and maintaining its firmness. Type III collagen accounts for about 10%-15% of the total collagen in the skin, providing the skin with suppleness and smoothness.

[0003] Prolonged exposure to ultraviolet (UV) radiation leads to the generation of large amounts of reactive oxygen species (ROS), activating numerous cytokines and affecting collagen production. On the other hand, it increases the quantity and activity of micronutrients (MMPs), promoting collagen degradation. When collagen fibers on the surface of fibroblasts break, the mechanosensitive forces on the fibroblasts weaken, causing them to reduce or lose their ability to synthesize collagen. This results in a decrease in collagen in the dermis, causing the skin to gradually lose elasticity, become loose, and eventually develop wrinkles. If lost collagen is replenished and the ability of fibroblasts in the dermis to synthesize collagen is restored through certain techniques, skin elasticity can be effectively restored, skin texture improved, and wrinkles reduced.

[0004] Collagen supplementation has gone through three stages: Phase 1: Animal-derived collagen It is mainly extracted from animal tissues, commonly from pig skin, cow Achilles tendon, and fish skin. Its sequence differs from human collagen, making it prone to causing rejection reactions when introduced into the body. It has poor water solubility, does not easily bind to the human body, and skin application can easily lead to allergic reactions.

[0005] Phase Two: Human-like Collagen Human-like collagen is a high-molecular-weight biological protein produced by reverse transcribing human collagen mRNA into cDNA, recombining a gene segment after enzymatic digestion into E. coli, and then processing it through high-density fermentation, separation, refolding, and purification. The main raw materials are glucose and inorganic salts. It is a collagen-mimicking product made from glucose and inorganic salts. While human-like collagen is relatively close to human collagen, it is more likely to trigger an immune response in the human body, has lower biological activity, lower genetic stability, retains some chemical substances, and carries the risk of rejection.

[0006] Phase 3: Humanized Collagen Based on the original gene sequence of human skin type III collagen, the highly water-soluble and bioactive portions were optimized and recombined using codon optimization to obtain a novel recombinant humanized type III collagen sequence. This sequence is then mass-produced using bio-fermentation technology. It is 100% identical to the human collagen gene sequence, causes no immune rejection or allergic reactions, possesses the functions of human collagen, and is easily absorbed by the human body.

[0007] However, these three stages of collagen supplementation only serve a filling function, have a short duration in the body, and are easily eliminated. They cannot cross-link with mature collagen fibers already present in the body, cannot bind to integrin receptors on fibroblasts, and cannot restore the collagen synthesis capacity of fibroblasts. Therefore, a new collagen supplementation method needs to be developed that allows the supplemented collagen to cross-link with mature collagen fibers while simultaneously restoring the collagen synthesis capacity of fibroblasts.

[0008] With the successful expression of mRNA as a novel therapeutic drug in various species and the validation of its safety during the COVID-19 pandemic, the potential of mRNA as a nucleic acid drug has become more apparent. Compared to traditional drugs, mRNA has several advantages. First, mRNA technology utilizes the body's own translation mechanism to express target proteins, ensuring consistency with pre-existing human proteins and avoiding the immunogenicity associated with proteins produced using other expression systems, such as CHO, thus guaranteeing the safety of the drug molecule. Second, the production of mRNA drug molecules can be achieved through in vitro enzymatic transcription (IVT) technology, eliminating the need for cell expansion and avoiding complex processes such as cell culture and purification, simplifying the production process and making product quality easier to control. However, the inherent instability and negative charge of mRNA molecules hinder their direct use, requiring the use of other carriers for delivery.

[0009] LNP, short for Lipid Nanoparticle, is a lipid vesicle with a uniform lipid core, typically around 100 nm in diameter. This unique structure and size endow it with distinctive physical and chemical properties, enabling it to effectively encapsulate and protect nucleic acid drugs, such as mRNA, siRNA, and antisense oligonucleotides, for delivery to target tissues or cells via the bloodstream. Due to its unique delivery advantages, such as high delivery efficiency, high biocompatibility, strong loading capacity, and rapid preparation process, it has been widely used for mRNA drug delivery.

[0010] To effectively replenish lost collagen in the skin and restore the collagen synthesis capacity of fibroblasts, we use lipid nanoparticles. Using LNP as a carrier, mRNA encoding type III collagen is precisely delivered into skin cells. This efficiently expresses the corresponding collagen, enabling it to exert its biological functions, restore skin quality, and slow down the aging process. Summary of the Invention

[0011] The purpose of this invention is to provide an mRNA and its application in delaying skin aging. The mRNA is loaded into lipid nanoparticles to deliver type III collagen mRNA into skin cells, enabling it to be expressed endogenously within the cells, thereby increasing the collagen content in the dermis, restoring skin quality, and delaying aging.

[0012] To achieve the above objectives, the present invention provides an mRNA, which is a type III collagen mRNA, with the sequence shown in SEQ ID NO: 1-6.

[0013] This invention also provides the application of the above-mentioned mRNA in delaying aging.

[0014] Furthermore, it can be applied to increase the expression level of collagen in the dermis.

[0015] Furthermore, it can be applied to the endogenous expression of collagen, thereby increasing collagen content.

[0016] The present invention also provides an anti-aging formulation comprising the above-mentioned mRNA loaded within lipid nanoparticles (LNPs).

[0017] Furthermore, the formulation also contains recombinant collagen.

[0018] This invention also provides the application of the above-mentioned formulation in increasing collagen expression levels.

[0019] The present invention also provides the application of the above-mentioned formulation in the endogenous expression of collagen and the increase of collagen content.

[0020] The advantages and positive effects of the mRNA described in this invention and its application in improving skin quality and delaying aging are as follows: 1. This invention optimizes the COL3A1-mRNA sequence to improve the expression level and stability of collagen. At the same time, it loads the mRNA into lipid nanoparticles, enabling the mRNA drug to endogenously express collagen, significantly increasing the collagen content and thus delaying aging.

[0021] 2. This invention uses LNP to encapsulate the target mRNA fragment, which can effectively and continuously derive the target protein (collagen), exhibiting high efficiency and scalability.

[0022] 3. This invention uses LNPs to encapsulate the target mRNA fragment. LNPs have the characteristics of high nucleic acid encapsulation rate, effective cell transfection, strong tissue penetration, and low cytotoxicity and immunogenicity, which are more conducive to drug delivery. This allows mRNA drugs to be delivered to cells safely and efficiently without entering the cell nucleus, protecting them from degradation and avoiding the safety risks of genome integration and cell transformation. Attached Figure Description

[0023] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0024] Figure 1 The following are the characterization results of LNPs in this embodiment of the invention, where A is the particle size of SEQ ID NO: 1 LNP, B is the particle size of SEQ ID NO: 2 LNP, and C is the particle size of LgBit LNP; Figure 2 The following are the characterization results of LNP in the embodiments of the present invention, where A is the LNP Zeta potential of SEQ ID NO: 1, B is the LNP Zeta potential of SEQ ID NO: 2, and C is the LgBit LNP Zeta potential; Figure 3 The expression levels of the optimized COL3A1-mRNA protein (SEQ ID NO: 1-6) in HEK293 cells are shown in this embodiment of the invention. Figure 4 The images shown are of the optimized COL3A1-mRNA protein (SEQ ID NO: 1-6) in vivo in animals, as described in this embodiment of the invention. Figure 5The results show the expression levels of the COL3A1-mRNA optimized protein (SEQ ID NO: 1-6) in animals in this embodiment of the invention. Detailed Implementation

[0025] Hereinafter, exemplary embodiments according to this application will be described in detail with reference to the accompanying drawings. Obviously, the described embodiments are merely a part of the embodiments of this application, and not all of the embodiments of this application. It should be understood that this application is not limited to the exemplary embodiments described herein. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without inventive effort are within the scope of protection of this invention.

[0026] Furthermore, unless otherwise defined, the technical or scientific terms used in this invention shall have the ordinary meaning as understood by one of ordinary skill in the art to which this invention pertains.

[0027] Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without inventive effort are within the scope of protection of this invention. Experimental methods in the following embodiments that do not specify specific conditions are generally determined according to national standards. Experimental instruments, equipment, and reagents in the following embodiments that do not specify their sources are all commercially available materials.

[0028] Unless otherwise defined or stated, all technical and scientific terms used in this invention have the same meaning as those skilled in the art. Furthermore, any methods and materials similar to or equivalent to those described herein may be applied to the methods of this invention.

[0029] Example 1: Preparation and Characterization of LNP (1) LNP preparation: Wild-type type III collagen, optimized type III collagen mRNA (C-terminally fused to a Hibit tag), and LgBit solution were thawed on ice. The mRNA was diluted with 50 mM citrate buffer (pH=4) to prepare the aqueous phase. The molar ratio of each LNP component was Lipid5:DOPE:cholestorol:DMG-PEG2000 38.4:12.3:47.4:1.9, and mixed in anhydrous ethanol to prepare the organic phase. The aqueous and organic phase solutions were drawn into 3 ml syringes, and the air in the syringes was expelled. The syringe containing the mRNA solution was inserted into the "C" inlet, and the syringe containing the mixed lipids was inserted into the "R" inlet. The aqueous / organic phase flow rate ratio (FRR) was set to 3:1. After running the device, the sample was collected. The collected solution was placed in an ultrafiltration tube, centrifuged and concentrated, and finally the LNP buffer was replaced with 20 mM Tris-HCl (pH 7.4) buffer.

[0030] (2) LNP characterization: For each type of LNP, 5 μL of ultrafiltration-concentrated LNP was added to 1 mL of PBS and placed in a cuvette. The particle size (PDI) of the LNP was measured using a Litesizer 100 particle size analyzer; the zeta potential of the LNP was measured using a zeta potential analyzer.

[0031] LNP particle size and zeta potential results Figure 1 As shown Example 2: In vitro expression verification (1) Cell plating Two flasks containing HEK239 T25 cells that had reached 90% confluence were removed from the incubator. The culture medium was discarded, and 3 mL of PBS was added. The flasks were gently shaken to wash away any remaining culture medium. After discarding the PBS, 1 mL of trypsin was added for digestion. The flasks were gently shaken to allow the trypsin to infiltrate the entire cell mass, and then the trypsin was immediately aspirated. Under a microscope, once the cells became rounded, 3 mL of DMEM high-glucose medium containing 10% FBS (without antibiotics) was added. The flask walls were gently pipetted to collect the cells. The two flasks of cells were then merged, and 1 mL of cells were transferred to each well of a 12-well plate. The plates were incubated at 37°C in a 5% CO2 incubator. Cell growth was observed, and transfection was performed when the cell confluence reached 70-80%.

[0032] (2) Cell transfection Before transfection, replace the culture medium in the 12-well plate with new 10% FBS DMEM high-glucose medium (without antibiotics).

[0033] Using serum-free DMEM high-glucose medium, add the corresponding type III collagen mRNA-LNP dropwise at a rate of 0.5 μg / well and mix gently. Protein expression levels are detected using the Nano-Glo® HiBiT kit after 24 hours.

[0034] The expression level of type III collagen in HEK293 cells is as follows: Figure 2 As shown. Experimental results show that the optimized SEQ ID:02 mRNA has a higher expression level in vitro than the WT sequence.

[0035] Example 3: In vivo expression verification Three BALB / c-Nude mice (1822g each) were used as a group. Each mouse received an intradermal injection of 0.1ml of the test solution. Twenty-four hours later, D-Luciferin substrate imaging was performed. Before imaging, each animal was intraperitoneally injected with 0.2ml of a 15mg / mL D-Luciferin fluorescent substrate solution and anesthetized with isoflurane. The mice were placed on the imaging stage in a supine position with their mouth and nose aligned with the glass tube opening, limbs exposed, and tails moved into the imaging area. A maximum of three mice were placed at a time for imaging, and the mice must not touch each other. The imaging mode was Luminescent. After imaging, the batch processing option was selected to import the images to be processed. In the Bioluminescence imaging detection mode, the unit needed to be adjusted to Radiance (Photons). The region to be analyzed was selected manually or automatically. "Measure ROIs" was then clicked to export the data in CSV format.

[0036] Experimental Groups: Group sequence test sample Dosage (μg / each) 1PBS-2WT / LNP +Lgbit / LNP5+13SEQ ID:01 / LNP +Lgbit / LNP5+14SEQID:02 / LNP +Lgbit / LNP5+15SEQ ID:03 / LNP +Lgbit / LNP5+16SEQ ID:04 / LNP +Lgbit / LNP5+17SEQ ID:05 / LNP +Lgbit / LNP5+18SEQ ID:06 / LNP +Lgbit / LNP5+1 Experimental results: SEQ ID:02 and SEQ ID:06 showed the highest expression levels in mice, which were higher than those of the WT sequence, and were basically consistent with the in vitro expression results.

[0037] Therefore, this invention utilizes the aforementioned mRNA and its application in anti-wrinkle and skin quality improvement. The mRNA is loaded into lipid nanoparticles, so that the mRNA drug does not need to enter the cell nucleus and is free from degradation, allowing it to be delivered to cells safely and efficiently. It also endogenously expresses collagen, increases collagen content, and thus has the effects of anti-wrinkle and improving skin quality.

[0038] The various embodiments of this disclosure have been described above. These descriptions are exemplary and not exhaustive, nor are they limited to the disclosed embodiments. Many modifications and variations will be apparent to those skilled in the art without departing from the scope and spirit of the described embodiments. The terminology used herein is chosen to best explain the principles, practical application, or improvement of the technology in the market, or to enable others skilled in the art to understand the embodiments disclosed herein.

Claims

1. A collagen type III (Col3A1) mRNA molecule, characterized in that, The Col3A1 mRNA molecule comprises a CDS coding region encoding Col3A1), which is selected from the nucleotide sequences shown in SEQ ID NO: 1-6 or nucleotide sequences having codon degeneracy and having at least 90% identity to the nucleotide sequences shown in SEQ ID NO: 1-6.

2. The Col3A1 mRNA molecule of claim 1, characterized in that, The mRNA is modified by one or more of N6-methyladenosine (m6A), 5-methylcytosine (m5C), 1-methyladenosine (m1A), 7-methylguanosine (m7G), pseudouridine (Ψ) or 4-acetylcytosine (ac4C).

3. The Col3A1 mRNA molecule of claim 1, wherein, The mRNA molecule is composed of three mRNA sequences respectively, which are marked as: F1, F2, F3.

4. The F1 fragment of claim 2 is a nucleotide sequence shown in SEQ ID NO: 7-12, the F2 fragment is: SEQ ID NO: 13, and the F3 fragment is: SEQ ID NO: 14-19.

5. A plasmid, characterized in that, The collagen type III (Col3A1) mRNA molecule of any one of claims 1-4.

6. A host cell, characterized in that, The mRNA molecule of any one of claims 1-4 or the plasmid of claim 5.

7. An mRNA pharmaceutical composition, characterized in that, The mRNA pharmaceutical composition comprises the collagen type III (Col3A1) mRNA molecule of any one of claims 1-4.

8. An mRNA drug, characterized in that The mRNA pharmaceutical comprises the mRNA pharmaceutical composition of claim 7 and a drug delivery carrier selected from one or more of a liposome nanoparticle (LNP), a cationic lipid complex, a polymeric nanoparticle, a protamine nanoparticle, a polypeptide nanoparticle, a viral vector, an inorganic nanoparticle, a cell-penetrating peptide, or a self-assembling RNA-lipid complex.

9. A method for the preparation of the mRNA drug of claim 8, characterized in that, The preparation method comprises the following steps: (1) Dissolve the cationic lipid, the auxiliary phospholipid, the cholesterol and the pegylated phospholipid in an organic solvent according to a certain molar ratio to obtain a mixture A of lipid nanoparticles; wherein the cationic lipid is Lipid 5, the auxiliary phospholipid is DOPE, and the pegylated phospholipid is DMG-PEG2000; (2) Dilute the mRNA to obtain a mixture B; (3) Mix the mixture A and the mixture B to prepare the mRNA pharmaceutical.

10. A collagen type III Col3A1 mRNA molecule, characterized in that, The collagen type III (Col3A1) mRNA molecule is obtained by the preparation method of claim 9.

11. Use of the mRNA pharmaceutical composition of claim 7, the mRNA pharmaceutical of claim 8, the mRNA pharmaceutical prepared by the preparation method of claim 9, or the collagen type III (Col3A1) mRNA molecule of claim 10 in the preparation of a drug for ultraviolet protection, skin whitening, skin elasticity, skin quality improvement, scar removal, stretch mark removal, and wound (including trauma) repair.

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