CN115992146A - Modified mRNA molecules and related products and uses - Google Patents

Abstract

The invention belongs to the technical field of biological medicine, and particularly relates to a modified mRNA molecule and application thereof. The modified mRNA molecule comprising a coding region encoding IGF1, wherein at least one uracil in the modified mRNA molecule is replaced with N ¹ -methyl pseudouracil. The invention also provides IGF1 gene modified mesenchymal stem cells transfected with said modified mRNA fractionAnd (5) a seed. The synthesized modified mRNA molecules and IGF1 gene modified mesenchymal stem cells can promote the healing of corneal wound surfaces, promote the formation of corneal nerves, promote the proliferation of limbal stem cells, the migration of limbal stem cells and the like, and can be used as a treatment means for damaging cornea to play a role in tissue multidimensional repair and regeneration.

Description

Modified mRNA molecules and related products and uses

Technical Field

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

Background Art

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.

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.

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.

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 protein delivery based on mRNA 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 chemical 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.

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

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.

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

The first aspect of the present invention protects a modified mRNA molecule, wherein 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.

In certain embodiments, 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.

In certain embodiments, the modified mRNA comprises N ¹ -methylpseudouracil in:

IGF1 coding region;

or, IGF1 coding region and 5′-UTR;

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

In certain embodiments, uracil (U) in the 5'-UTR, IGF1 coding region, and 3'-UTR are all replaced with N ¹ -methylpseudouracil (m1Ψ).

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

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.

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

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

In certain embodiments, the 3'-UTR comprises a 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.

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

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

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.

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

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

1) A sequence as shown in SEQ ID No. 6, wherein all uracils are replaced by N ¹ -methyl pseudouracil;

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.

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

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

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

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.

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.

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.

Preferably, the mesenchymal stem cells are adipose-derived mesenchymal stem cells.

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.

In certain embodiments, the acceptable excipients include carriers, media, such as sterile water or physiological 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 physiological saline, isotonic solutions containing glucose or other auxiliary drugs, such as D-sorbitol, D-mannose, D-mannitol, sodium chloride, appropriate solubilizers such as alcohols (such as ethanol), polyols (propylene glycol, PEG, etc.), non-ionic surfactants (Tween 80, HCO-50), etc. can be used in combination.

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.

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

In certain embodiments, the pharmaceutical composition is mainly directed to mammals. The mammals are preferably rodents, even-toed ungulates, perissodactyls, lagomorphs, primates, etc. The primates are preferably monkeys, apes or humans.

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:

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

2) Preparation of products for promoting corneal wound healing;

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

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

5) preparing products for promoting limbal stem cell migration;

6) Prepare products for inhibiting corneal angiogenesis.

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

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

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

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. It can promote the multi-faceted repair of corneal damage caused by alkali burns in animal models.

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

Figure 1 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 = 50 μm; Figure B is a statistical graph of transfection efficiency 24 hours after transfection by flow cytometry analysis; Figure C is a statistical graph of the average fluorescence intensity 24 hours after transfection by flow cytometry analysis; Figure D is a statistical graph of IGF1 mRNA expression level 24 hours 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 transfected with modGFP; ADSC ^modIGF1 , 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 < 0.0001.

Figure 2 shows that the subconjunctival injection of ADSC ^modIGF1 in Example 1 of the present invention can achieve rapid repair of alkali-burned cornea. Figure A is a representative slit-light 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 comparative statistical chart of corneal sodium fluorescein grading. CTR, corneal alkali burn control group with subconjunctival injection of PBS; ADSC ^modLuc , ADSC ^modLuc group injected subconjunctivally after corneal alkali burn modeling; ADSC ^modIGF1 , ADSC ^modIGF1 group injected subconjunctivally after corneal alkali burn modeling. 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.

Figure 3 shows that ADSC ^modIGF1 in Example 2 of the present invention is beneficial for the multi-dimensional repair and regeneration of damaged cornea. Figure A is a representative image of corneal H&E staining and Masson's trichrome staining at D16 after alkali burn in each group of mice; Figure B is a statistical analysis of the corneal thickness ratio based on the results of histological staining; Figure C is a statistical analysis of the ratio of corneal epithelial thickness; Figure D is a representative image of the whole cornea stained with Tubulinβ-Ⅲ in each group, as well as the peripheral and central corneal nerve fiber images of each group; Figure E is an analysis and statistical chart of the percentage of the area occupied by the subbasal nerve. CTR, corneal alkali burn control group; ADSC ^modLuc , subconjunctival injection of ADSC ^modLuc group after corneal alkali burn modeling; ADSC ^modIGF1 , subconjunctival injection of ADSC ^modIGF1 group after corneal alkali burn modeling. 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.

Figure 4 shows that ADSC ^modIGF1 in Example 3 of the present invention promotes 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 a representative immunofluorescence staining image of Ki67 in LSCs after 24 hours of co-culture, with DAPI counterstained nuclei, scale bar = 20μm; Figure C shows a representative image of LSCs after migration obtained by Transwell migration experiment after 24 hours of co-culture with different groups of ADSCs, scale bar = 50μm; Figure D is a comparative statistical chart 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. 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.

DETAILED DESCRIPTION

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.

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.

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.

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.

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

To achieve the above-mentioned purpose, the present invention also provides mesenchymal stem cells (ADSC ^modIGF1 ) that overexpress IGF1 modified mRNA (IGF1 modRNA).

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.

The present invention uses ADSCs (ADSC ^modIGF1 ) that overexpress IGF1 modified mRNA to treat corneal damage, corneal nerve loss, limbal stem cell loss and other problems caused by alkali burns. The damaged cornea repair effect of ^{ADSC modIGF1} is mainly achieved by promoting corneal nerve regeneration and regulating the fate of limbal stem cells.

One of the objectives of the present invention is to provide a modified mRNA molecule, wherein 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 finds that mRNA with uracil replaced by ^N1 -methylpseudouracil (m1Ψ) has much better performance than other modified bases, can achieve high expression and effective translation of mRNA, increase the stability of mRNA molecules, and avoid immune response and cytotoxicity caused by mRNA introduction into cells.

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.

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:

IGF1 coding region;

or, IGF1 coding region and 5′-UTR;

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

More preferably, the uracil (U) in the 5'-cap structure, 5'-UTR, IGF1 coding region, and 3'-UTR are all modified with N ¹ -methylpseudouracil (m1Ψ).

In the present invention, the 5' cap structure has m ⁷ G ^5' ppp ^5' N (m), wherein 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 recruiting mRNA precursor splicing, polyadenylation and nuclear export. In a preferred embodiment, it is m ⁷ G ^5' pppNm (Cap1).

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 axonemal 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.

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.

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, 110 to 135 nucleotides, or 125 to 150 nucleotides. In a preferred embodiment, it is 120 nucleotides.

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

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

1) A sequence as shown in SEQ ID No. 6, wherein all uracils are replaced by N ¹ -methyl pseudouracil;

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.

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.

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 level 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.

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:

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

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

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 pharmaceutically acceptable excipients.

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.

The present invention prepares ADSCs (ADSC ^modIGF1 ) overexpressing IGF1 protein by transfecting IGF1 modified mRNA, and then injects the ADSCs into the subconjunctiva of mice for the treatment of acute corneal alkali burns.

In the present invention, through the construction of a mouse corneal alkali burn model and continuous clinical evaluation, it was shown that ADSC ^modIGF1 treatment can effectively promote the transparency and thickness of the damaged cornea to reach normal levels and accelerate the healing rate of corneal epithelial defects, indicating that overexpression of IGF1 can indeed improve the in vivo efficacy of ADSCs.

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 corneas. The corneas of mice in the ADSC ^modIGF1 group have more complete epithelium, a more compact and orderly arranged matrix, and the distribution of corneal nerves is more dense.

In the present invention, limbal stem cells (LSCs) were co-cultured with ADSCs modified by modRNA, and the results showed that ADSC ^modIGF1 could significantly enhance the proliferation and migration ability of LSCs.

The innovative therapeutic product ADSC ^modIGF1 , which combines modRNA technology with stem cell therapy, 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. It has been proven that ADSC ^modIGF1 combines the therapeutic advantages of ADSCs and IGF1, can effectively promote multi-dimensional repair of damaged corneas, and is a new therapeutic product with good potential for repairing acute alkali burns of the cornea.

Although the present invention uses a mouse corneal alkali burn model to explore and verify the therapeutic effect of ADSC ^modIGF1 , the damaged cornea repair effect of ADSC ^modIGF1 is not limited to this disease model. The cornea, as an important structural barrier of the eyeball, has the function of protecting the intraocular tissue from environmental factors, and the destruction of 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, although the causes are different, have similar pathological manifestations, such as persistent corneal epithelial defects, corneal neovascularization, corneal nerve damage and lack of limbal stem cells. In the present invention, ADSC ^modIGF1 combines the therapeutic advantages of ADSCs and IGF1 using modRNA technology, which 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.

Example 1

In this example, the DNA sequences of 5'UTR, 3'UTR and IGF1 in Table 1 were used as templates and modRNA was obtained by in vitro transcription.

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, in vitrotranscription) is shown in Table 1.

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

Table 1

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

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 by purifying RNA using an Ambion MEGAclear spin column and treating it 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 -methyl pseudouracil.

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.

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.

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

Table 2 DNA template sequence and modIGF1 sequence

Example 2

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

1. Isolation and culture of ADSCs

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.

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.

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.

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.

2. modRNA transfection

2.1 Methods

MessengerMAX^TMReagent转染1×10⁵个ADSCs。Use 2 μL per 1 μg of IGF1 modRNA

1×10 ⁵ ADSCs were transfected with MessengerMAX ^TM Reagent.

MessengerMAX^TMReagent分别在Opti-MEM(Invitrogen)中稀释，在室温(RT)下孵育5分钟。1) Combine IGF1 modRNA and transfection reagent

MessengerMAX ^™ Reagent was diluted in Opti-MEM (Invitrogen) and incubated at room temperature (RT) for 5 minutes.

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

3) 4 h after transfection of modRNA-lipid complex into ADSCs cells, 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 ADSC ^modIGF1 until the culture medium was analyzed or the cells were collected for subsequent experiments.

modIGF1 transfection group: ADSC ^modIGF1 . At the same time, a modGFP transfection group (ADSC ^modGFP ) and a blank control group (CTR) were set up. After the GFP modRNA in Example 1 was transfected into ADSCs, ADSC ^modGFP was formed; the blank control group (CTR) was ADSCs that were not transfected with any gene.

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). The results are shown in Figure 1, B and C.

After transfection, photos were taken for 1-6 consecutive days to detect its expression in cells. The results are shown in Figure 1A.

2.2 Results

As shown in Figure 1A, 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.

As shown in Figure 1B and C, the transfection efficiency detected by flow cytometry can reach more than 90% within 24 hours, and the average fluorescence intensity can reach more than 1.8×10 ⁶ .

3. Detection

3.1 Real-time quantitative PCR

3.1.1 Methods

Total RNA was extracted from ADSC ^modIGF1 in step 2 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 manufacturer's instructions.

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.

3.1.2 Results

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

3.2 Enzyme-linked immunosorbent assay (ELISA)

3.2.1 Methods

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.

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.

3.2.2 Results

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

As can be seen from Figure 1E, 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.

Example 3

In this example, ADSC ^modLuc and ADSC ^modIGF1 obtained in Example 2 were transformed into a mouse corneal alkali burn model, and the clinical evaluation of corneal injury, corneal tissue morphology, limbal stem cells (LSCs) proliferation and limbal stem cells (LSCs) migration were investigated. The following are included:

The Luc modRNA in Example 1 was transfected into ADSCs using the same method as that used in Example 2 to prepare ADSC ^modIGF1 , thereby forming an ADSC ^modLuc group.

Isolation and culture of LSCs

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.

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.

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

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.

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.

1. Establishment of mouse corneal alkali burn model

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.

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

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.

Subconjunctival injection of ADSCs

Mice with corneal alkali burn model were randomly divided into control group (CTR), ADSC ^modLuc group and ADSC ^modIGF1 group.

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

ADSC ^modLuc group: 1×10 ⁵ ADSC ^{modLuc cells} obtained in Example 2 dissolved in 10 μL PBS were injected subconjunctiva of corneal alkali burn model mice using a 50 μL syringe combined with a 33-gauge metal needle (Hamilton Company, Reno, NV, USA).

ADSC ^modIGF1 group: 1×10 ⁵ ADSC ^modIGF1 cells obtained in Example 2 dissolved in 10 μL PBS were injected subconjunctiva of corneal alkali burn model mice using a 50 μL syringe combined with a 33-gauge metal needle.

After subconjunctival injection, the mice were continuously clinically evaluated for corneal damage until day 16, and corneal histological examination and analysis were performed subsequently.

2. Research and Analysis

2.1 Clinical evaluation of corneal injury

2.1.1 Methods

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

Corneal opacity was evaluated using a slit lamp microscope (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 points. The results are shown in Figure 2, A and B.

0 = Completely clear

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

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

3 = Iris is severely opaque and pupil is barely visible

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

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 and photographed under cobalt blue light. The results are shown in Figure 2, C and D.

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

2.1.2 Results

As shown in Figure 2A and B, after continuous examination, the results of slit lamp microscopy showed that subconjunctival injection of ADSC ^modIGF1 cells could significantly alleviate corneal edema caused by alkali burns and quickly restore the transparency of the damaged cornea to normal levels.

As can be seen from Figure 2C and D, the sodium fluorescein staining images showed that both the ADSC ^modLuc and ADSC ^modIGF1 groups could effectively accelerate wound healing, but the degree of corneal epithelial defect in the ADSC ^modIGF1 group was significantly lower than that in the other two groups.

2.2 Analysis and research on corneal tissue morphology

2.2.1 Methods

The mice were sacrificed on day 16, and the eyeballs were embedded in paraffin and cut into 6 μm sections. Subsequently, the sections were 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 3A.

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, B and C.

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

2.2.2 Results

As shown in Figure 3A, 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, manifested by abnormal cell morphology and arrangement of the corneal epithelium, and loose and disordered corneal stromal fibers; the corneal epithelium of the ADSCs-treated group, especially the ADSC ^modIGF1 group, was thicker and relatively normal, and the stromal layer was dense and orderly.

As can be seen from Figure 3B and C, based on the results of histological staining, the corneal thickness and corneal epithelial thickness of each group were measured and analyzed, and it was found that the cornea of the ADSC ^modIGF1 group was thinner, the epithelial layer was thicker, and the corneal morphology was relatively normal.

As can be seen from Figure 3D and E, after Tubulinβ-Ⅲ staining, the representative images of the peripheral and central fibers of the cornea respectively showed that ADSC ^modIGF1 cell therapy undoubtedly played an important role in promoting the regeneration and repair of alkali-burned corneal nerve fibers.

As shown in Figure 3D, the nerve fiber density in the peripheral and central areas of the cornea in the ADSC ^modIGF1 cell-treated group was greater than that in the other two groups.

2.3 Analysis of the proliferation of limbal stem cells (LSCs)

2.3.1 Methods

After LSCs were co-cultured with modRNA-modified ADSCs (ADSC ^modLuc and ADSC ^modIGF1 ) for 24 hours, they were seeded in 24-well plates. There were three groups: LSC ^ADSC-modluc group co-cultured with LSCs and ADSC ^modLuc ; LSC ^{ADSC- modIGF1} group co-cultured with LSCs and ADSC modIGF1; and CTR group.

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.

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

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.

Total RNA was extracted from the cells cultured for 12 h in step 2.3.1 of this embodiment 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 relative expression was determined using the 2-ΔΔCT method. The results are shown in Figure 4 A.

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 4B.

2.3.2 Results

As shown in Figure 4A, co-culture with ADSCs can promote the proliferation of LSCs, and from the Ki67 gene expression level, it can be seen that co-culture of LSCs with ADSCs transfected with modIGF1 (LSC ^ADSC-modIGF1 group) can significantly promote cell proliferation more than the control group.

As shown in Figure 4B, the Ki67 immunocytochemical staining results of LSCs in each group were consistent with the RT-PCR results, suggesting that ADSC ^modIGF1 treatment was most beneficial for promoting LSC proliferation.

2.4 Analysis of the migration of limbal stem cells (LSCs)

2.4.1 Methods

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

To detect migration ability, a 24-well polycarbonate membrane cell culture chamber with a pore size of 8 μm (SPL LifeSciences Co., Ltd., Korea) was used. 2×10 ³ cells/well of 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 ADSC ^modIGF1 ) on the migration ability of LSCs.

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, and the results are shown in Figure 4C and D.

2.4.2 Results

As shown in Figure 4C and D, both ADSC ^modLuc and ADSC ^modIGF1 can promote the migration of LSCs, but the effect is more significant when co-cultured with ADSC ^modIGF1 .

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, it can achieve pluripotency in somatic cell reprogramming. The present invention combines modRNA technology with cell therapy to obtain ADSCs (ADSC ^modIGF1 ) that overexpress IGF1. Experiments have shown that it can significantly promote the morphological and functional recovery of the cornea of alkali burn model mice. In addition to the epithelial and matrix morphology of the cornea 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, so it has good clinical application prospects.

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 ideas disclosed by the present invention shall still be covered by the claims of the present invention.

Claims (10)

1. A modified mRNA molecule, wherein the modified mRNA molecule comprises a coding region encoding IGF1, and wherein at least one uracil in the modified mRNA molecule is replaced with N ¹ -methyl pseudouracil.

2. The modified mRNA molecule of claim 1, wherein said modified mRNA molecule comprises a 5 '-cap structure, a 5' -UTR, an IGF1 coding region, a 3'-UTR, a 3' poly a tail in the 5 'to 3' direction.

3. The modified mRNA molecule of claim 1 or 2, wherein the modified mRNA comprises N in ¹ -methyl pseudouracil:

IGF1 coding region;

alternatively, IGF1 coding region and 5' -UTR;

alternatively, IGF1 coding region, 5'-UTR and 3' -UTR.

4. The modified mRNA molecule of claim 2, wherein the 5'-UTR comprises a 5' -UTR of an a gene selected from one of a β -globin (HBB) gene, a heat shock protein 70 (Hsp 70) gene, an axin heavy chain 2 (DNAH 2) gene, and a 17β -hydroxysteroid dehydrogenase 4 (HSD 17B 4) gene, or a homolog, fragment, or variant thereof; preferably, the DNA coding sequence corresponding to the 5' -UTR comprises a sequence as shown in SEQ ID No. 1;

And/or the 3'-UTR comprises a 3' -UTR of a B gene or a homologue, fragment or variant thereof, the B gene being selected from one of an Albumin (ALB) gene, an alpha-globin gene, a beta-globin (HBB) gene, a tyrosine hydroxylase gene, a heat shock protein 70 (Hsp 70) gene, a lipoxygenase gene and a collagen alpha gene; preferably, the DNA coding sequence corresponding to the 3' -UTR comprises a sequence as shown in SEQ ID No. 2;

and/or the length of the polyA tail is 100-150 nucleotides;

and/or the DNA coding sequence corresponding to the IGF1 coding region comprises a sequence shown as SEQ ID No. 3.

5. The modified mRNA molecule of claim 1, comprising at least one of:

1) As shown in SEQ ID No.6, and wherein uracil is replaced with N ¹ -a sequence of methyl pseudouracil;

2) A sequence which encodes a protein of the same sequence as the nucleotide sequence of 1) but which differs from the nucleotide sequence of 1) by the degeneracy of the genetic code.

6. A biological material related to the modified mRNA molecule of any one of claims 1-5, comprising at least one of:

1) A polynucleotide encoding the modified mRNA molecule of any one of claims 1-5;

2) A recombinant expression vector comprising the polynucleotide of 1).

7. An IGF1 genetically modified mesenchymal stem cell, characterized in that the IGF1 genetically modified mesenchymal stem cell overexpresses the IGF1 gene, preferably obtained by transfection of a modified mRNA molecule according to any one of claims 1-5.

8. The IGF1 genetically modified mesenchymal stem cell of claim 7, wherein the mesenchymal stem cell is selected from one or more of an adipose-derived mesenchymal stem cell, a bone marrow-derived mesenchymal stem cell, an umbilical cord-derived mesenchymal stem cell, a placenta-derived mesenchymal stem cell, a cornea stroma-derived mesenchymal stem cell, and a limbal-derived mesenchymal stem cell.

9. A pharmaceutical composition comprising a modified mRNA molecule according to any one of claims 1-5 or a biological material according to claim 6 or an IGF1 genetically modified mesenchymal stem cell according to claim 7 or 8.

10. Use of a modified mRNA molecule according to any of claims 1 to 5 or a biological material according to claim 6 or a mesenchymal stem cell of an IGF1 modification gene according to claim 7 or 8 or a pharmaceutical composition according to claim 9,

Comprising the use in at least one of:

1) Preparing a product for preventing and/or treating corneal injury or corneal disease;

2) Preparing a product for promoting the healing of the cornea wound surface;

3) Preparing a product that promotes corneal nerve fiber formation;

4) Preparing a product for promoting proliferation of limbal stem cells;

5) Preparing a product that promotes limbal stem cell migration;

6) Preparing the product for inhibiting cornea angiogenesis.

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