Modified mRNA molecules and related products and uses

Abstract

The present invention belongs to the field of biomedicine technology, and specifically relates to a modified mRNA molecule and its use. The modified mRNA molecule comprises a coding region encoding IGF1, and at least one uracil in the modified mRNA molecule is replaced by N 1 ‑methyl pseudouracil. The present invention also provides IGF1 gene-modified mesenchymal stem cells, which are transfected with the modified mRNA molecules. The synthetic modified mRNA molecules and IGF1 gene-modified mesenchymal stem cells of the present invention can promote corneal wound healing, corneal nerve formation, limbal stem cell proliferation and limbal stem cell migration, etc., and can be used as a treatment method for damaged cornea to exert a multi-dimensional tissue repair and regeneration effect.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to modified mRNA molecules and related products and uses. Background Art

[0002] The cornea is the most important refractive medium in the eye and is of great significance to the formation of good vision. As an important structural barrier, the cornea can protect the intraocular tissue from external environmental factors, but due to its special position, it is extremely susceptible to various types of injuries such as chemical injuries and thermal burns. Among them, corneal alkali burns, due to the strong lipid solubility of alkaline chemicals, sustainable penetration and damage to corneal tissue, cause a severe inflammatory response, lead to the loss of limbal stem cells and severe damage to corneal tissue, often cause corneal dissolution or even perforation, and are the main cause of blindness worldwide.

[0003] Although the current clinical treatment methods for corneal alkali injury vary, mainly to inhibit excessive inflammatory response and promote the repair of corneal epithelium, few methods can achieve comprehensive therapeutic effects. For example, corticosteroid eye drops play an important role in inhibiting inflammation, but long-term use may cause ocular complications, damage corneal integrity, and even lead to corneal melting. Amniotic membrane transplantation is widely used in clinical practice due to its good anti-inflammatory effect, but the rapid dissolution and peeling of amniotic membrane limits corneal repair to a certain extent. In the late stage of corneal injury, allogeneic corneal transplantation is generally used to partially restore the structure and function of the cornea, but corneal donor resources are limited, and postoperative rejection often leads to poor long-term prognosis. Therefore, the repair process of corneal injury is multidimensional and complex, and it is urgent to explore effective and comprehensive new treatment methods.

[0004] In recent years, mesenchymal stem / stromal cells (MSCs) have played an important role in tissue engineering and regenerative medicine research due to their unique biological characteristics and potential clinical application value. MSCs have important immunomodulatory and anti-inflammatory properties. Subconjunctival injection of MSCs in animals with corneal alkali burns is believed to promote corneal wound healing and inhibit stromal inflammation, neovascularization and fibrosis. Among MSCs, adipose-derived mesenchymal stem cells (ADSCs) are favored due to their wide range of tissue sources and low immunogenicity. Although MSCs have been proven to be effective in the treatment of corneal alkali burns, natural mesenchymal stem cells are limited by their poor tissue specificity and lesion targeting. Repairing alkali-burned corneas requires not only general anti-inflammatory treatment, but also comprehensive repair of various tissues including corneal epithelium, stroma, nerves, and even limbus, which is difficult to achieve through simple MSCs treatment. Corneal nerves and limbal stem cells (LSCs) are important factors in the comprehensive recovery of corneal morphology and function, and the effects of MSCs on these two are rarely reported. How to modify MSCs to improve their efficacy, thereby more effectively reducing the progression of acute damage and avoiding late surgical treatment, is a direction worth studying.

[0005] In order to improve the therapeutic effect, MSCs are often genetically modified by exogenous gene transfection according to the characteristics of the disease to enhance their specific functions. It is worth noting that mRNA-based protein delivery has been widely used in the field of regenerative medicine. In order to improve the stability of mRNA and eliminate the risk of insertion mutations, synthetic chemically modified mRNA (modRNA) has received widespread attention. As a new and efficient gene delivery vector and nucleic acid therapy, modRNA can achieve pluripotency in somatic cell reprogramming due to its strong transient gene expression, minimal immunogenicity, and efficient and dose-controlled protein delivery. In our previous study, by combining modRNA technology with cell therapy, it was verified that cell-mediated modRNA delivery may be an alternative treatment strategy for lower limb ischemia, bone defects or fat transplantation, proving the feasibility of the cell-based modified mRNAs delivery system.

[0006] In this study, insulin-like growth factor-1 (IGF1), a member of the insulin family, was used as a target gene to improve the intrinsic repair ability of MSCs for damaged cornea. IGF1 is a multifunctional cytokine with a wide range of biological activities, playing an important role in maintaining and regulating cell growth, proliferation, differentiation, maturation and regeneration. In addition, IGF1 is an important neurotrophic factor that promotes nerve regeneration and repair after peripheral nerve injury. In ophthalmology, IGF1 and its receptors have been shown to be expressed on the surface of human corneal and conjunctival epithelial cells. However, there are no reports on the application and therapeutic mechanism of IGF1 modRNA on the ocular surface. Summary of the invention

[0007] In view of the above-mentioned shortcomings of the prior art, the object of the present invention is to provide modified mRNA molecules and related products and uses, in order to solve the problems existing in the prior art.

[0008] To achieve the above purpose, the present invention specifically adopts the following technical solutions.

[0009] The first aspect of the present invention protects a modified mRNA molecule, wherein the modified mRNA molecule comprises a coding region encoding IGF1, wherein at least one uracil in the modified mRNA molecule is replaced by N 1 -Methylpseudouracil.

[0010] In certain embodiments, the synthetic IGF1-mRNA molecule comprises, from 5' to 3' direction, a 5'-cap structure, a 5'-UTR, an IGF1 coding region, a 3'-UTR, and a 3' PolyA tail.

[0011] In certain embodiments, the modified mRNA comprises N 1 -Methylpseudouracil:

[0012] IGF1 coding region;

[0013] or, IGF1 coding region and 5′-UTR;

[0014] or, IGF1 coding region, 5'-UTR and 3'-UTR.

[0015] In some embodiments, the uracil (U) in the 5'-UTR, IGF1 coding region, and 3'-UTR is replaced by N 1 -Methylpseudouracil (m1Ψ).

[0016] In certain embodiments, the 5' cap structure has m 7 G5'ppp5'N(m), where m 7G is N7-methylguanosine; p is phosphate; ppp is triphosphate; N is any nucleoside, such as adenine (A), guanosine (G), cytidine (C) and uridine (U), or other naturally occurring nucleosides or modified nucleosides.

[0017] In certain embodiments, the 5'-UTR comprises the 5'-UTR of an A gene or a homolog, fragment or variant thereof, wherein the A gene is selected from the group consisting of a β-globin (HBB) gene, a heat shock protein 70 (Hsp70) gene, axonemal dynein heavy chain 2 (DNAH2) gene and a 17β-hydroxysteroid dehydrogenase 4 (HSD17B4) gene. For example, the variant sequence may have at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with the wild-type 5'-UTR sequence of the corresponding gene.

[0018] Preferably, the 5'-UTR is the 5'-UTR of the 17β-hydroxysteroid dehydrogenase 4 (HSD17B4) gene or a homolog, fragment or variant thereof.

[0019] More preferably, the DNA coding sequence corresponding to the 5'-UTR comprises the sequence shown in SEQ ID NO.1.

[0020] In certain embodiments, the 3'-UTR comprises the 3'-UTR of a B gene or a homolog, fragment or variant thereof, wherein the B gene is selected from one of an albumin (ALB) gene, an α-globin gene, a β-globin (HBB) gene, a tyrosine hydroxylase gene, a heat shock protein 70 (Hsp70) gene, a lipoxygenase gene and a collagen α gene. For example, the variant sequence may have at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with the wild-type 3'-UTR sequence of the corresponding gene.

[0021] More preferably, the DNA coding sequence corresponding to the 3'-UTR comprises the sequence shown in SEQ ID No.2.

[0022] In certain embodiments, the polyA tail has a length of 100 to 150 nucleotides.

[0023] Preferably, the length of the polyA tail may be 100 to 125 nucleotides, 110 to 135 nucleotides, or 125 to 150 nucleotides. In a preferred embodiment, the length is 120 nucleotides.

[0024] In certain embodiments, the DNA coding sequence corresponding to the IGF1 coding region comprises the sequence shown in SEQ ID No.3.

[0025] In certain embodiments, at least one of the following is included:

[0026] 1) The sequence shown in SEQ ID No.6, wherein uracil is replaced by N 1 -Sequence of methyl pseudouracil;

[0027] 2) A sequence that encodes a protein of the same sequence as the nucleotide sequence in 1), but is different from the nucleotide sequence in 1) due to the degeneracy of the genetic code.

[0028] The second aspect of the present invention protects the biological material associated with the modified mRNA molecule as described above, including at least one of the following:

[0029] 1) A polynucleotide encoding a modified mRNA molecule as described above;

[0030] 2) A recombinant expression vector containing the polynucleotide described in 1).

[0031] The third aspect of the present invention protects an IGF1 gene-modified mesenchymal stem cell, wherein the IGF1 gene-modified mesenchymal stem cell overexpresses the IGF1 gene, preferably, obtained by transfecting the modified mRNA molecule as described above.

[0032] In the present invention, after the modified mRNA molecules are transfected into mesenchymal stem cells, the transfection efficiency is high and high expression of the IGF1 gene can be achieved.

[0033] In certain embodiments, the mesenchymal stem cells are selected from one or more of adipose-derived mesenchymal stem cells, bone marrow-derived mesenchymal stem cells, umbilical cord-derived mesenchymal stem cells, placenta-derived mesenchymal stem cells, corneal stroma-derived mesenchymal stem cells, and limbus-derived mesenchymal stem cells.

[0034] Preferably, the mesenchymal stem cells are adipose-derived mesenchymal stem cells.

[0035] The fourth aspect of the present invention protects a pharmaceutical composition comprising the modified mRNA molecule as described above, or the biomaterial as described above, or the IGF1 gene-modified mesenchymal stem cells as described above, and a pharmaceutically acceptable excipient.

[0036] In certain embodiments, the acceptable excipients include carriers, media, such as sterile water or saline, stabilizers, excipients, antioxidants (such as ascorbic acid), buffers (phosphoric acid, citric acid, other organic acids, etc.), preservatives, surfactants (PEG, Tween, etc.), chelating agents (EDTA, etc.), adhesives, etc. In addition, other low molecular weight polypeptides; proteins such as serum albumin, gelatin or immunoglobulin; amino acids such as glycine, glutamine, asparagine, arginine and lysine; sugars or carbohydrates such as polysaccharides and monosaccharides; sugar alcohols such as mannitol or sorbitol. When preparing aqueous solutions for injection, such as saline, isotonic solutions containing glucose or other auxiliary drugs, such as D-sorbitol, D-mannose, D-mannitol, sodium chloride, appropriate solubilizers such as alcohols (ethanol, etc.), polyols (propylene glycol, PEG, etc.), non-ionic surfactants (Tween 80, HCO-50), etc. can be used in combination.

[0037] In certain embodiments, the pharmaceutical composition can be adapted to any form of administration, which can be oral, nasal, rectal, intravenous, parenteral, etc. The pharmaceutical composition can be prepared into injections, sterile powders for injection, tablets, pills, capsules, lozenges, elixirs, powders, granules, syrups, solutions, tinctures, aerosols, powder sprays, or suppositories.

[0038] In certain embodiments, the pharmaceutical composition may also be used in combination with other treatment methods, including surgery, radiotherapy, chemotherapy, and targeted therapy.

[0039] In certain embodiments, the pharmaceutical composition is mainly directed to mammals. The mammals are preferably rodents, artiodactyls, perissodactyls, lagomorphs, primates, etc. The primates are preferably monkeys, apes or humans.

[0040] The fifth aspect of the present invention protects the use of the modified mRNA molecule as described above, or the biomaterial as described above, or the IGF1 gene-modified mesenchymal stem cell as described above, or the pharmaceutical composition as described above, including at least one of the following uses:

[0041] 1) Preparation of products for preventing and / or treating corneal damage or corneal diseases;

[0042] 2) Preparation of products for promoting corneal wound healing;

[0043] 3) preparing products for promoting the formation of corneal nerve fibers;

[0044] 4) preparing products for promoting the proliferation of limbal stem cells;

[0045] 5) preparing products for promoting limbal stem cell migration;

[0046] 6) Prepare products for inhibiting corneal angiogenesis.

[0047] In certain embodiments, the corneal injury comprises a chemical burn or a thermal burn.

[0048] In certain embodiments, the corneal disease comprises exposure keratopathy or neurotrophic keratitis.

[0049] Compared with the prior art, the present invention has the following beneficial effects:

[0050] 1) This technology has invented a suitable therapeutic product ADSC modIGF1 , which is based on mesenchymal stem cell therapy and combines modRNA technology to enhance its therapeutic advantages, can promote multifaceted repair of corneal damage caused by alkali burns in animal models.

[0051] 2) Mesenchymal stem cells overexpressing modIGF1 can promote the repair and regeneration of corneal nerves and regulate the fate of limbal stem cells. They can be used as a treatment for damaged cornea to play a multi-dimensional tissue repair and regeneration role. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] Figure 1 It shows that high-efficiency transfection of modRNA can be achieved in ADSCs in Example 1 of the present invention. Figure A is a representative fluorescence image of the daily GFP signal dynamics of ADSCs from D1 to D6 after transfection, scale bar = 50μm; Figure B is a statistical graph of transfection efficiency 24h after transfection by flow cytometry analysis; Figure C is a statistical graph of the average fluorescence intensity 24h after transfection by flow cytometry analysis; Figure D is a statistical graph of IGF1 mRNA expression level 24h after transfection; Figure E is a statistical graph of newly generated IGF1 protein in the culture medium detected by ELISA after modIGF1 was transfected into ADSCs. CTR, normal ADSCs control group; ADSC modGFP , ADSCs group transfected with modGFP; ADSC modIGF1 , ADSCs group transfected with modIGF1. Data are presented as mean ± standard deviation; * indicates that there is a statistical difference between the two groups, p < 0.05; **** indicates that there is a statistical difference between the two groups, p < 0.0001.

[0053] Figure 2 Shown is the subconjunctival injection of ADSCs in Example 1 of the present invention modIGF1It can achieve rapid repair of alkali-burned cornea. Figure A is a representative slit-lamp microscopic examination image of the cornea, showing the corneal opacity at D0, D4, D8, and D16 after alkali burn; Figure B is a statistical chart of clinical scoring analysis of corneal transparency at different time points; Figure C is a representative sodium fluorescein staining image, showing the degree of corneal epithelial defect at D0, D4, D8, and D16 after alkali burn; Figure D is a statistical chart of the analysis and comparison of corneal sodium fluorescein grading. CTR, corneal alkali burn control group with subconjunctival injection of PBS; ADSC modLuc , ADSCs were injected subconjunctivally after corneal alkali burn modeling modLuc Group; ADSC modIGF1 , ADSCs were injected subconjunctivally after corneal alkali burn modeling modIGF1 The data are presented as mean ± standard deviation; * indicates that there is a statistical difference between the two groups, p < 0.05; ** indicates that there is a statistical difference between the two groups, p < 0.01; *** indicates that there is a statistical difference between the two groups, p < 0.001; **** indicates that there is a statistical difference between the two groups, p < 0.0001.

[0054] Figure 3 Shown as ADSC in Example 2 of the present invention modIGF1 It is conducive to the multi-dimensional repair and regeneration of damaged cornea. Figure A shows the representative images of corneal H&E staining and Masson's trichrome staining at D16 after alkali burn in each group of mice; Figure B shows the statistical analysis of corneal thickness ratio based on histological staining results; Figure C shows the statistical analysis of corneal epithelial thickness ratio; Figure D shows the representative images of the whole cornea stained with Tubulinβ-Ⅲ in each group, as well as the peripheral and central corneal nerve fiber images; Figure E shows the statistical analysis of the percentage of the area occupied by the subbasal nerve. CTR, corneal alkali burn control group; ADSC modLuc , ADSCs were injected subconjunctivally after corneal alkali burn modeling modLuc Group; ADSC modIGF1 , ADSCs were injected subconjunctivally after corneal alkali burn modeling modIGF1 The data are presented as mean ± standard deviation; * indicates that there is a statistical difference between the two groups, p < 0.05; ** indicates that there is a statistical difference between the two groups, p < 0.01; *** indicates that there is a statistical difference between the two groups, p < 0.001; **** indicates that there is a statistical difference between the two groups, p < 0.0001.

[0055] Figure 4 Shown is ADSC in Example 3 of the present invention modIGF1Promote the proliferation and migration of limbal stem cells in vitro. Figure A shows the expression level of Ki67 mRNA in LSCs after 24 hours of co-culture with ADSCs transfected with modRNA; Figure B shows the representative immunofluorescence staining image of Ki67 in LSCs after 24 hours of co-culture, with DAPI counterstained cell nuclei, scale bar = 20μm; Figure C shows the representative image of LSCs after migration obtained by Transwell migration assay after 24 hours of co-culture with different groups of ADSCs, scale bar = 50μm; Figure D shows the comparative statistical graph of the relative number of cell migration after crystal violet staining. CTR, normal LSCs control group; LSC ADSC -modLuc , LSCs group co-cultured with ADSCs transfected with modLuc; LSC ADSC -modIGF1, LSCs group co-cultured with ADSCs transfected with modIGF1. Data are presented as mean ± standard deviation; * indicates that there is a statistical difference between the two groups, p < 0.05; ** indicates that there is a statistical difference between the two groups, p < 0.01; *** indicates that there is a statistical difference between the two groups, p < 0.001; **** indicates that there is a statistical difference between the two groups, p < 0.0001. DETAILED DESCRIPTION

[0056] The following is a description of the implementation of the present invention by means of specific embodiments. Those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification.

[0057] Before further describing the specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terms used in the examples of the present invention are intended to describe specific embodiments, rather than to limit the scope of protection of the present invention. The test methods in the following examples without specifying specific conditions are generally carried out under conventional conditions or under conditions recommended by the manufacturers.

[0058] When the embodiments give numerical ranges, it should be understood that, unless otherwise specified in the present invention, both endpoints of each numerical range and any numerical value between the two endpoints can be selected. Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those generally understood by those skilled in the art. In addition to the specific methods, equipment, and materials used in the embodiments, according to the grasp of the prior art by those skilled in the art and the record of the present invention, any methods, equipment, and materials of the prior art similar or equivalent to the methods, equipment, and materials described in the embodiments of the present invention can also be used to realize the present invention.

[0059] At present, the clinical treatment methods for corneal alkali damage are mainly to inhibit excessive inflammatory response and promote the repair of corneal epithelium, but few methods can achieve comprehensive therapeutic effects. The repair process of corneal damage is multidimensional and complex, and it is urgent to explore effective and comprehensive new treatment methods.

[0060] For the above purpose, the present invention provides a modified mRNA molecule, which is an IGF1 modified mRNA (IGF1modRNA) that overexpresses IGF1.

[0061] For the above purpose, the present invention also provides mesenchymal stem cells (ADSC) that overexpress IGF1 modified mRNA (IGF1 modRNA) modIGF1 ).

[0062] For the above purpose, the present invention also provides use of mesenchymal stem cells overexpressing IGF1 modified mRNA (IGF1 modRNA) in products for preventing and / or treating corneal damage or corneal diseases.

[0063] The present invention is to overexpress IGF1 modified mRNA in ADSCs (ADSC modIGF1 ) is used to treat corneal damage caused by alkali burns, corneal nerve defects, and limbal stem cell loss. modIGF1 The damaged cornea repair effect is mainly achieved by promoting corneal nerve regeneration and regulating the fate of limbal stem cells.

[0064] One of the objects of the present invention is to provide a modified mRNA molecule, wherein the modified mRNA molecule comprises a coding region encoding IGF1, wherein at least one uracil in the modified mRNA molecule is replaced by N 1 -Methylpseudouracil.

[0065] The present invention finds that, through N 1 -Methyl pseudouracil (m1Ψ) replaces uracil in mRNA, which has much better performance than other modified bases. It can achieve high expression and effective translation of mRNA, increase the stability of mRNA molecules, and avoid immune response and cytotoxicity caused by the introduction of mRNA into cells.

[0066] The present invention finds that gene delivery using modified mRNA molecules (IGF1 modRNA) does not carry the risk of adverse and potentially harmful chromosomal integration; mRNA delivery is more efficient than DNA transfection in terms of both the total amount of cell uptake and the number of target cells; and mRNA directs protein expression almost immediately after reaching the cytoplasm.

[0067] In the present invention, the synthetic IGF1-mRNA molecule comprises a 5'-cap structure, a 5'-UTR, an IGF1 coding region, a 3'-UTR, and a 3'PolyA tail from the 5' to the 3' direction. Preferably, the modified mRNA comprises modified nucleotides in the following parts:

[0068] IGF1 coding region;

[0069] or, IGF1 coding region and 5′-UTR;

[0070] or, IGF1 coding region, 5'-UTR and 3'-UTR.

[0071] More preferably, the uracil (U) in the 5'-cap structure, 5'-UTR, IGF1 coding region, and 3'-UTR are all replaced by N 1 -Methyl pseudouracil modification (m1Ψ).

[0072] In the present invention, the 5' cap structure has m 7 G 5’ ppp 5’ N(m), where m 7 G is 7-methylguanosine; p is phosphate; ppp is triphosphate; m is methyl; N is any nucleoside, such as adenine (A), guanosine (G), cytosine (C) and uridine (U), or other naturally occurring nucleosides or modified nucleosides. The 5' cap structure is crucial for the efficient translation of mRNA. mRNA without a cap structure cannot be translated and is immunogenic. The 5' cap structure can protect mRNA from exonuclease cleavage and is the only recognition anchor point for protein factors that recruit mRNA precursor splicing, polyadenylation and nuclear export. In a preferred embodiment, m 7 G 5’ pppNm(Cap1).

[0073] In the present invention, the 5'-UTR comprises the 5'-UTR of the A gene or its homologue, fragment or variant, and the A gene is selected from the β-globin (HBB) gene, the heat shock protein 70 (Hsp70) gene, the axoneme dynein heavy chain 2 (DNAH2) gene and the 17β-hydroxysteroid dehydrogenase 4 (HSD17B4) gene. For example, the variant sequence may have at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with the wild-type 5'-UTR sequence of the corresponding gene. Preferably, the 5'-UTR is the 5'-UTR of the 17β-hydroxysteroid dehydrogenase 4 (HSD17B4) gene or its homologue, fragment or variant. The 5'-UTR sequence is most critical for protein expression. In a preferred embodiment, the DNA coding sequence corresponding to the 5'-UTR comprises the sequence shown in SEQ ID NO.1.

[0074] In the present invention, the 3'-UTR comprises the 3'-UTR of the B gene or its homolog, fragment or variant, and the B gene is selected from one of the albumin (ALB) gene, α-globin gene, β-globin (HBB) gene, tyrosine hydroxylase gene, heat shock protein 70 (Hsp70) gene, lipoxygenase gene and collagen α gene. For example, the variant sequence can have at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with the wild-type 3'-UTR sequence of the corresponding gene. 3'-UTR is a key driving force for the half-life of mRNA. Most cis-acting elements that affect the stability of mRNA are usually located in 3'-UTR. In a preferred embodiment, the DNA coding sequence corresponding to the 3'-UTR comprises a sequence as shown in SEQID No.2.

[0075] In the present invention, the length of the polyA tail is 100 to 150 nucleotides. Preferably, the length of the polyA tail can be 100 to 125 nucleotides, or 110 to 135 nucleotides, or 125 to 150 nucleotides. In a preferred embodiment, it is 120 nucleotides.

[0076] In the present invention, the DNA coding sequence corresponding to the IGF1 coding region comprises the sequence shown in SEQ ID No.3.

[0077] In the present invention, the modified mRNA molecule includes at least one of the following:

[0078] 1) As shown in SEQ ID No.6, wherein uracil is replaced by N 1 -Sequence of methyl pseudouracil;

[0079] 2) A sequence that encodes a protein of the same sequence as the nucleotide sequence in 1), but is different from the nucleotide sequence in 1) due to the degeneracy of the genetic code.

[0080] A second object of the present invention is to provide an IGF1 gene-modified mesenchymal stem cell, wherein the IGF1 gene-modified mesenchymal stem cell overexpresses the IGF1 gene, preferably, obtained by transfecting the modified mRNA molecule as described above.

[0081] The present invention finds that mesenchymal stem cells (ADSCs) can tolerate liposome-mediated IGF1 modRNA transfection. Flow cytometry detection and fluorescence microscopy observation show that IGF1 modRNA transfection of ADSCs can reach a peak expression in a very short time, and the transfection efficiency is high. Real-time quantitative PCR detection and enzyme-linked immunosorbent assay (ELISA) detection show that the secreted IGF1 expression and protein level are significantly increased, indicating that ADSCs can exert their effects through the large-scale secretion of IGF1.

[0082] The third object of the present invention is to provide a biological material related to the modified mRNA molecule as described above, comprising at least one of the following:

[0083] 1) A polynucleotide encoding a modified mRNA molecule as described above;

[0084] 2) A recombinant expression vector containing the polynucleotide described in 1).

[0085] A fourth object of the present invention is to provide a pharmaceutical composition comprising the modified mRNA molecule as described above, or the biomaterial as described above, or the IGF1 gene-modified mesenchymal stem cells as described above, and a pharmaceutically acceptable excipient.

[0086] The fifth object of the present invention is to provide the use of the modified mRNA molecule as described above, or the biomaterial as described above, or the mesenchymal stem cell modified by the IGF1 gene as described above, or the pharmaceutical composition as described above, including at least one of the following uses: 1) preparing a product for preventing and / or treating corneal damage or corneal disease; 2) preparing a product for promoting corneal wound healing; 3) preparing a product for promoting corneal nerve fiber formation; 4) preparing a product for promoting the proliferation of limbal stem cells; 5) preparing a product for promoting the migration of limbal stem cells; 6) preparing a product for inhibiting corneal angiogenesis. In some embodiments, the corneal damage includes chemical burns and thermal burns. In some embodiments, the corneal disease includes exposure keratopathy or neurotrophic keratitis.

[0087] The present invention prepares ADSCs (ADSCs) overexpressing IGF1 protein by transfecting IGF1 modified mRNAmodIGF1 ), which was then injected into the subconjunctiva of mice for the treatment of acute corneal alkali burns.

[0088] In the present invention, the construction of a mouse corneal alkali burn model and continuous clinical evaluation showed that ADSCs modIGF1 The treatment can effectively promote the transparency and thickness of the damaged cornea to normal levels and accelerate the healing of corneal epithelial defects, indicating that overexpression of IGF1 can indeed improve the in vivo efficacy of ADSCs.

[0089] In the present invention, the results of histochemical staining showed that ADSCs can indeed promote corneal wound healing, but ADSCs transfected with modIGF1 have better ability to repair damaged cornea. modIGF1 The corneas of the mice in the control group had more complete epithelium, a more compact and orderly arranged stroma, and a denser distribution of corneal nerves.

[0090] In the present invention, the co-culture of limbal stem cells (LSCs) and modRNA-modified ADSCs showed that ADSCs modIGF1 It can significantly enhance the proliferation and migration ability of LSCs.

[0091] The present invention combines modRNA technology with stem cell therapy to create an innovative therapeutic product ADSC modIGF1 , can be used for multi-dimensional regeneration and repair of damaged corneas. While achieving corneal tissue morphology repair, it promotes the regeneration of corneal nerve fibers and the fate regulation of limbal stem cells, and has good clinical application prospects. modIGF1 It combines the therapeutic advantages of ADSCs and IGF1 and can effectively promote the multi-dimensional repair of damaged cornea. It is a new therapeutic product with good potential for repairing acute alkali burn cornea.

[0092] Although the present invention used the mouse corneal alkali burn model to carry out ADSC modIGF1 Exploration and verification of therapeutic effects, but ADSC modIGF1 The repair effect of damaged cornea is not limited to this disease model. The cornea, as an important structural barrier of the eyeball, has the function of protecting intraocular tissues from environmental factors, and damage to the corneal structure, transparency and function can easily lead to severe vision loss. Clinically, a variety of corneal injuries or diseases, including chemical injuries, thermal burns, exposure keratopathy or neurotrophic keratitis, have similar pathological manifestations despite different causes, such as persistent corneal epithelial defects, corneal neovascularization, corneal nerve damage and limbal stem cell deficiency. In the present invention, ADSC modIGF1Combining the therapeutic advantages of ADSCs and IGF1 using modRNA technology can efficiently and comprehensively promote the repair and regeneration of various parts of the corneal tissue structure, including the corneal epithelium, stroma and nerves, and has potential therapeutic value for the treatment of clinical corneal alkali burns and other corneal injuries or diseases with similar pathological characteristics.

[0093] Example 1

[0094] In this example, the DNA sequences of 5'UTR, 3'UTR and IGF1 in Table 1 were used as templates to obtain modRNA by in vitro transcription.

[0095] In vitro transcription was performed using an RNA transcription kit (T7 High yield RNA Transcription kit, purchased from Novoprotein), which contains T7 RNA polymerase. The reaction system for in vitro transcription (IVT) is shown in Table 1.

[0096] The T7 kit for Nearshore Protein, a 20μL system, can usually produce 120-140μg of mRNA.

[0097] Table 1

[0098] RNA free water 6μL 10×buffer 2μL Cap1 0.75μL 100mM GTP 0.75μL 100mM ATP 1.5μL 100mM CTP 1.5μL 100mM N1UTP 1.5μL 100mM Linearized DNA template 5μL (PCR product concentration 200ng / μL) T7 RNA Enzyme 1μL Total 20μL

[0099] The reaction system in Table 1 was placed in a PCR instrument and reacted at 37°C for 3 hours, then 1 μL DNase I (RNase-free) was added, mixed thoroughly, and reacted at 37°C for 30 minutes. RNA was synthesized using an Ambion MEGAclear spin column to purify RNA and treated with Antarctic Phosphatase (New England Biolabs) at 37°C for 30 minutes to remove residual 5'-phosphates to obtain modified mRNA molecules (IGF1 modRNA). During the in vitro transcription synthesis process, uracil was replaced by N 1 -Methylpseudouracil.

[0100] The purity and concentration of the synthetic IGF1-mRNA were assessed using a Nanodrop spectrophotometer (Thermo Fisher Scientific, Waltham, MA) (concentration > 1 μg / μL, OD260 / 280 = 1.8-2.2), and the modified mRNA molecules (IGF1modRNA) were resuspended in 10 mM Tris HCl and 1 mM EDTA at a concentration of 1 μg / μL for later use.

[0101] At the same time, a GFP modRNA control group and a Luc modRNA control group were set up; the GFP modRNA control group was obtained by replacing the IGF1 coding sequence with the GFP coding sequence; the IGF1 coding sequence was replaced with the Luciferase coding sequence to obtain the Luc modRNA control group. The GFP coding sequence and the Luciferase coding sequence are shown in SEQ ID No. 4 and SEQ ID No. 5 in Table 1.

[0102] IGF1 modRNA, Luc modRNA and GFP modRNA are collectively referred to as modRNA.

[0103] Table 2 DNA template sequence and modIGF1 sequence

[0104]

[0105]

[0106]

[0107] Example 2

[0108] In this example, the modRNA synthesized in Example 1 was transfected into mesenchymal stem cells, including the following:

[0109] 1. Isolation and culture of ADSCs

[0110] Adipose tissue was collected from healthy female patients (20-30 years old) who underwent blepharoplasty for cosmetic purposes. This study was approved by the Medical Ethics Committee of the Ninth People's Hospital affiliated to Shanghai Jiao Tong University School of Medicine.

[0111] The surgery was performed by full incision blepharoplasty, and the removed adipose tissue fragments were cut into small pieces, digested with 0.1% type A collagenase (Roche, Manheim, Germany) at 37°C for 8 hours, and then centrifuged at 1500 rpm for 10 minutes.

[0112] The pelleted cells were resuspended in DMEM (HyClone, GE Healthcare, Little Chalfont, UK) medium supplemented with 10% fetal bovine serum (FBS; Invitrogen, California, USA) and 1% penicillin-streptomycin antibiotics (Gibco, Carlsbad, CA, USA) and cultured at 37°C.

[0113] The cells were cultured in a culture medium every 2 days and subcultured using TrypLE (Gibco) when the cells reached 80-90% density. All studies were performed using ADSCs between passages 3 and 5.

[0114] 2. modRNA transfection

[0115] 2.1 Methods

[0116] Use 2 μL per 1 μg of IGF1 modRNA MessengerMAX TM Reagent transfection 1×10 5 ADSCs.

[0117] 1) Combine IGF1 modRNA and transfection reagent MessengerMAX TM Reagents were diluted in Opti-MEM (Invitrogen) and incubated at room temperature (RT) for 5 minutes.

[0118] 2) The two mixtures were mixed together and incubated for 15 minutes to generate modRNA-lipid complexes.

[0119] 3) After transfection of modRNA-lipid complex into ADSCs cells for 4 h, the medium was completely replaced with DMEM (HyClone) supplemented with 10% FBS (Invitrogen) and 1% penicillin-streptomycin antibiotics (Gibco), and further cultured at 37°C to obtain ADSCs. modIGF1 , until the culture medium is analyzed or cells are collected for subsequent experiments.

[0120] modIGF1 transfection group: ADSC modIGF1 At the same time, a modGFP transfection group (ADSC modGFP ) and a blank control group (CTR). After the GFP modRNA in Example 1 was transfected into ADSCs, ADSCs were formed. modGFP ; The blank control group (CTR) was ADSCs without any gene transfection.

[0121] 24 hours after transfection, the transfection efficiency and mean fluorescence intensity of GFP modRNA were detected using a C6 flow cytometer (Beckman Coulter, CA, USA). Figure 1 Medium B and C.

[0122] After transfection, take pictures for 1-6 consecutive days to detect its expression in cells. The results are shown in Figure 1 Middle A.

[0123] 2.2 Results

[0124] from Figure 1 As shown in Figure A, primary human ADSCs were cultured in vitro and transfected with modGFP. Continuous observation under a fluorescence microscope showed that GFP was expressed in the cells for more than 6 days. Lipofectamine-mediated modRNA transfection was well tolerated in ADSCs and could achieve strong transient high gene expression in the cells.

[0125] from Figure 1 As shown in B and C, the transfection efficiency can reach more than 90% after 24 h detection by flow cytometry, and the average fluorescence intensity can reach 1.8×10 6 above.

[0126] 3. Detection

[0127] 3.1 Real-time quantitative PCR

[0128] 3.1.1 Methods

[0129] The ADSCs in step 2 were purified using the EZ-press RNA purification kit (EZ Bioscience, MN, USA). modIGF1 Total RNA was extracted from

[0130] Reverse transcription was performed using the PrimeScript RT kit (Takara Bio Inc., Otsu, Japan) according to the manufacturer's instructions.

[0131] PCR detection was performed using Power SYBR Green PCR Master Mix (Applied Biosystems, Foster, CA, USA) on a 7500 Real-Time PCR Detection System (Thermo Fisher Scientific), and the relative expression level was determined using the 2-ΔΔCT method.

[0132] 3.1.2 Results

[0133] from Figure 1 As shown in D, by RT-PCR, the expression of IGF1 in the ADSCs group transfected with modIGF1 increased by about 8000 times 24 hours after transfection compared with the CTR control group and modGFP group.

[0134] 3.2 Enzyme-linked immunosorbent assay (ELISA)

[0135] 3.2.1 Methods

[0136] To quantify the expression kinetics of IGF1 after ADSCs transfection, supernatants were collected at different time points (1, 2, 3, 4, 5, and 6 days) after transfection, and IGF1 protein levels were detected using an ELISA kit (R&D Systems, Inc., Minneapolis, MN, USA) according to the manufacturer's instructions.

[0137] The optical density value of absorbance was measured on a microplate reader (ELX800, BioTek, USA), and the protein content was determined using a standard curve.

[0138] 3.2.2 Results

[0139] For 6 consecutive days, the concentration of newly produced IGF1 protein in the culture medium was detected using an ELISA kit.

[0140] from Figure 1 As shown in Figure E, compared with the CTR control group and the modGFP transfection control group, the IGF1 protein level in the modIGF1 transfection group was significantly increased, especially on the first day after transfection.

[0141] Example 3

[0142] In this example, the ADSCs obtained in Example 2 were modLuc and ADSCs modIGF1 Transformed into the mouse corneal alkali burn model, the corneal injury clinical evaluation, corneal tissue morphology, limbal stem cells (LSCs) proliferation and limbal stem cells (LSCs) migration were investigated. The following are included:

[0143] The Luc modRNA in Example 1 was used to prepare ADSCs in the same manner as in Example 2. modIGF1 The same method was used to transfect ADSCs to form ADSCs modLuc Group.

[0144] Isolation and culture of LSCs

[0145] With the approval of the Eye Bank and Medical Ethics Committee of the Ninth People's Hospital affiliated to Shanghai Jiao Tong University School of Medicine, the remaining donor corneal rings after keratoplasty were collected.

[0146] The obtained corneal ring tissue was incubated with 10 mg / mL diapase II (Sigma-Aldrich, St. Louis, MO, USA) at 4°C for 8 h.

[0147] Subsequently, the epithelial cell sheets were separated from the matrix and digested with TrypLE at 37°C for 5 min.

[0148] The obtained single cell suspension was inoculated in DMEM / F12 medium (Invitrogen) containing 10% fetal bovine serum, 1% insulin-transferrin-selenium, 1% non-essential amino acids (Life Technologies, Carlsbad, CA), 0.4 μg / mL hydrocortisone (Wako, Osaka, Japan), 2 mM l-glutamine (Life Technologies), 10 ng / mL epidermal growth factor (Life Technologies), and 1% penicillin-streptomycin antibiotics.

[0149] The culture medium was changed every two days, and when LSCs reached 80-90% density, they were passaged using TrypLE. LSCs of passages 1 to 3 were used for subsequent experiments.

[0150] 1. Establishment of mouse corneal alkali burn model

[0151] All mouse experiments in this study were approved by the Medical Ethics Committee of the Ninth People's Hospital affiliated to Shanghai Jiao Tong University School of Medicine and were performed in accordance with the guidelines of the Chinese Animal Administration and the Association for Research in Vision and Ophthalmology (ARVO) Statement for the Use of Animals in Ophthalmic and Vision Research.

[0152] In this study, healthy 8-week-old female BALB / c mice were used to establish the corneal alkali burn model.

[0153] After the mice were anesthetized, oxybuprocaine hydrochloride eye drops (Santen Pharmaceutical Co., Ltd., Kita-ku, Osaka, Japan) were used for ocular surface anesthesia. Then, a round Whatman III filter paper soaked in 1.0N NaOH (Sigma–Aldrich, St. Louis, MO, USA) was placed on the central cornea of ​​the right eye of the mouse for 40 seconds, and then the damaged ocular surface was rinsed with PBS for 1 minute to obtain the mouse corneal alkali burn model.

[0154] Subconjunctival injection of ADSCs

[0155] Mice with corneal alkali burn model were randomly divided into control group (CTR), ADSC modLuc Groups and ADSC modIGF1 Group.

[0156] CTR group: 10 μL PBS was injected subconjunctivally in the corneal alkali burn model mice.

[0157] ADSC modLucGroup: 1×10 50 μL of PBS was injected into the conjunctiva of the corneal alkali burn model mice using a 50 μL syringe combined with a 33-gauge metal needle (Hamilton Company, Reno, NV, USA). 5 ADSCs obtained in Example 2 modLuc cell.

[0158] ADSC modIGF1 Group: 1×10 dapoxetine dissolved in 10 μL PBS was injected subconjunctivally into the corneal alkali burn model mice using a 50 μL syringe combined with a 33-gauge metal needle. 5 ADSCs obtained in Example 2 modIGF1 cell.

[0159] After administration via subconjunctival injection, mice were continuously clinically evaluated for corneal damage until day 16, and corneal histological examination and analysis were performed subsequently.

[0160] 2. Research and Analysis

[0161] 2.1 Clinical evaluation of corneal injury

[0162] 2.1.1 Methods

[0163] Mice in each group were clinically examined at designated times, and the researchers were unaware of the group assignment during scoring and data analysis.

[0164] The corneal opacity was evaluated by slit lamp microscopy (SL-D7, Topcon Inc., Tokyo, Japan) on days 0, 4, 8, and 16 after alkali burns, and the corneal edema score was recorded from 0 to 4. The results are shown in Figure 2 A and B.

[0165] 0 = Completely clear

[0166] 1 = slightly blurry, iris and pupil are easy to see

[0167] 2 = slightly opaque, iris and pupil can still be detected

[0168] 3 = Iris is severely opaque and pupil is barely visible

[0169] 4 = Totally opaque, no iris and / or pupil visible

[0170] Then, at 0, 4, 8, and 16 days after alkali burn, corneal fluorescein staining was performed with 2% sodium fluorescein (Sigma-Aldrich), and corneal epithelial defects were evaluated under cobalt blue light and photographed. The results are shown in Figure 2 Middle C and D.

[0171] Corneal epithelial defect scores were assessed and analyzed according to the National Eye Institute / Industry grading scale.

[0172] 2.1.2 Results

[0173] from Figure 2 As shown in Figures A and B, after continuous examination, slit lamp microscopy results showed that ADSCs were injected subconjunctivally. modIGF1 The cells can significantly relieve corneal edema caused by alkali burns and quickly restore the transparency of the damaged cornea to normal levels.

[0174] from Figure 2 As shown in C and D, the images of sodium fluorescein staining show that ADSC modLuc and ADSCs modIGF1 Both groups can effectively accelerate wound healing, but ADSC modIGF1 The degree of corneal epithelial defect in group A was significantly lower than that in the other two groups.

[0175] 2.2 Analysis and research on corneal tissue morphology

[0176] 2.2.1 Methods

[0177] The mice were sacrificed on day 16, and the eyeballs were embedded in paraffin and cut into 6 μm sections. The sections were then stained with H&E and Masson's trichrome (Sigma, St. Louis, MO, USA) according to the manufacturer's instructions. The staining results are shown in Figure 3 Middle A.

[0178] The stained sections were observed and photographed under a fluorescence microscope, and then the corneal / corneal epithelial thickness was analyzed using ImageJ. The results are shown in Figure 3 Medium B and C.

[0179] Corneal nerve staining: The whole corneas of each group were fixed in Zamboni fixative (Solarbio, Beijing, China) for 1 h, and then blocked in 0.1% Triton X-100, 2% goat serum, and 2% bovine serum albumin for 2 h. Subsequently, the blocked corneas were incubated with Tubulin β-Ⅲ antibody at 4 °C overnight, and then incubated with Alexa Fluor 488-conjugated secondary antibody at room temperature for 1 h. The incubated corneas were cut into petal shapes, and the corneal nerve morphology was observed and recorded using a fluorescence microscope. The results are shown in Figure 3 D. ImageJ was used to calculate the percentage of the area occupied by the nerve fibers under the cornea. The results are shown in Figure 3 Middle E.

[0180] 2.2.2 Results

[0181] from Figure 3As shown in Figure A, the results of hematoxylin & eosin (H&E) staining and Masson's staining showed that the tissue morphology of the CTR group was still poor 16 days after injury, showing abnormal cell morphology and arrangement of the corneal epithelium and loose and disordered corneal stromal fibers; the ADSCs-treated group, especially the ADSCs modIGF1 The corneal epithelium of the group was thicker and relatively normal, and the stroma was dense and orderly.

[0182] from Figure 3 As shown in B and C, based on the results of histological staining, the corneal thickness and corneal epithelial thickness of each group were measured and analyzed, and ADSC modIGF1 The cornea of ​​the first group was thinner, the epithelium was thicker, and the corneal morphology was relatively normal.

[0183] from Figure 3 As shown in D and E, representative images of the peripheral and central fibers of the cornea after Tubulin β-Ⅲ staining show that ADSC modIGF1 Cell therapy undoubtedly plays an important role in promoting the regeneration and repair of corneal nerve fibers injured by alkali.

[0184] from Figure 3 As shown in Figure 3, ADSC modIGF1 The density of nerve fibers in the peripheral and central areas of the cornea in the cell-treated group was greater than that in the other two groups.

[0185] 2.3 Analysis of the proliferation of limbal stem cells (LSCs)

[0186] 2.3.1 Methods

[0187] LSCs were combined with modRNA-modified ADSCs (ADSC modLuc and ADSCs modIGF1 ) After 24 hours of co-culture, the cells were seeded in a 24-well plate. There are three groups: LSCs and ADSCs modLuc Co-culture of LSCs ADSC-modluc Group; LSCs and ADSCs modIGF1 Co-culture of LSCs ADSC-modIGF1 group; and the CTR group.

[0188] After 12 h of culture, the cells were rinsed three times with PBS, fixed with 4% PFA for 15 min, permeabilized with 0.3% Triton X-100 (Sigma-Aldrich) diluted in PBS for 15 min, and blocked with 5% donkey serum (Sigma-Aldrich) at room temperature for 1 h.

[0189] Subsequently, cells were incubated with Ki67 antibody (Cell Signaling Technology, Inc. 3 Trask Lane Danvers, MA, USA) at 4°C overnight.

[0190] Then, the cells were incubated with Alexa fluorescent conjugated secondary antibody (BD Biosciences, San Jose, CA, USA) diluted 1:400 at room temperature for 1 h.

[0191] Total RNA was extracted from the cells cultured for 12 h in step 2.3.1 of this example using the EZ-press RNA purification kit (EZ Bioscience, MN, USA). Reverse transcription was performed using the PrimeScript RT kit (Takara Bio Inc., Otsu, Japan) according to the instructions. PCR detection was performed on a 7500 Real-Time PCR detection system (Thermo Fisher Scientific) using Power SYBR Green PCR Master Mix (Applied Biosystems, Foster, CA, USA), and the relative expression level was determined using the 2-ΔΔCT method. The results are shown in Figure 4 Middle A.

[0192] The cell nuclei were stained with DAPI (Invitrogen) for 10 min, and then observed and recorded with a fluorescence microscope (Nikon Eclipse 80i; Nikon Instruments, Tokyo, Japan), and the fluorescence intensity was analyzed with ImageJ. The results are shown in Figure 4 Middle B.

[0193] 2.3.2 Results

[0194] from Figure 4 As shown in Figure 5A, co-culture with ADSCs can promote the proliferation of LSCs, and the Ki67 gene expression level shows that LSCs and ADSCs transfected with modIGF1 (LSC ADSC-modIGF1 The co-culture of the two groups significantly promoted cell proliferation than that of the control group.

[0195] from Figure 4 As shown in Figure B, the Ki67 immunocytochemical staining results of LSCs in each group were consistent with the RT-PCR results, indicating that ADSCs modIGF1 treatment is most beneficial to promote LSC proliferation.

[0196] 2.4 Analysis of the migration of limbal stem cells (LSCs)

[0197] 2.4.1 Methods

[0198] Transwell assay was used to study the migration of LSCs under co-culture conditions.

[0199] To detect migration ability, a 24-well polycarbonate membrane cell culture chamber with a pore size of 8 μm (SPL Life Sciences Co., Ltd., Korea) was used. 3 LSCs were seeded in the upper chamber of the Transwell chamber, and ADSCs transfected with modRNA were seeded in the lower chamber to explore the effect of ADSCs transfected with modRNA (ADSC modLuc and ADSCs modIGF1 ) on the migration ability of LSCs.

[0200] After 24 hours of incubation, the liquid was discarded and the cells were fixed with 4% paraformaldehyde (PFA) at room temperature for 15 minutes. After staining with 0.1% crystal violet (Biyuntian Biotechnology Co., Ltd., China) at room temperature for 20 minutes, cells in three fields of view were randomly photographed and counted using a microscope. The results are shown in Figure 4 Middle C and D.

[0201] 2.4.2 Results

[0202] from Figure 4 As shown in C and D, ADSC modLuc and ADSCs modIGF1 Both can promote the migration of LSCs, but not ADSCs modIGF1 The co-culture effect is more significant.

[0203] Mesenchymal stem cells, cell carriers, play an important role in tissue engineering and regenerative medicine research because of their important immunomodulatory and anti-inflammatory properties. Many studies have confirmed that their subconjunctival injection can promote corneal wound healing, inhibit matrix inflammation, neovascularization and fibrosis. The modRNA technology can genetically modify mesenchymal stem cells to enhance their specific functions. Due to its strong transient gene expression, minimal immunogenicity, and efficient and dose-controlled protein provision, pluripotency can be achieved in somatic cell reprogramming. The present invention combines modRNA technology with cell therapy to obtain ADSCs (ADSCs) that overexpress IGF1. modIGF1 ), which has been proven by experiments to be able to significantly promote the morphological and functional recovery of the cornea of ​​mice with alkali burn models. In addition to the corneal epithelium and matrix morphology being closer to normal tissues, the corneal nerve fibers are also the densest, and the regeneration and repair of the cornea after injury is the best. Therefore, it has good prospects for clinical application.

[0204] The above embodiments are merely illustrative of the principles and effects of the present invention, and are not intended to limit the present invention. Anyone familiar with the art may modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by a person of ordinary skill in the art without departing from the spirit and technical concept disclosed by the present invention shall still be covered by the claims of the present invention.

Claims

1. Use of IGF1 gene-modified mesenchymal stem cells, wherein the IGF1 gene-modified mesenchymal stem cells overexpress the IGF1 gene; the IGF1 gene-modified mesenchymal stem cells are obtained by transfecting modified mRNA molecules, including at least one of the following uses: 1) Preparation of products for preventing and / or treating corneal damage; 2) Preparation of products for promoting corneal wound healing; The sequence of the modified mRNA molecule is shown in SEQ ID No.6, wherein uracil is replaced by N 1 -Methylpseudouracil.

2. The use according to claim 1, characterized in that The modified mRNA molecule is from 5 ’ To 3 ’ Direction contains 5'-cap structure, 5 ’ -UTR, IGF1 coding region, 3'-UTR, 3 ’ PolyA tail.

3. The use according to claim 1, characterized in that The mesenchymal stem cells are selected from one or more of adipose-derived mesenchymal stem cells, bone marrow-derived mesenchymal stem cells, umbilical cord-derived mesenchymal stem cells, placenta-derived mesenchymal stem cells, corneal stroma-derived mesenchymal stem cells, and limbus-derived mesenchymal stem cells.

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