An mRNA-polypeptide complex and its preparation method and application

Abstract

The present invention provides an mRNA-polypeptide complex and a preparation method and application thereof. The present invention synthesizes the Smad7-mRNA-polypeptide complex for the first time. The mRNA-polypeptide complex has the function of significantly delaying the aging of primary human trabecular meshwork cells, and can be used for the treatment of glaucoma. It also has the potential to be developed into an inhibitor of other aging diseases, such as neurodegenerative diseases, skin aging, premature aging of stem cells, premature aging of immune cells, etc.; the mRNA-polypeptide complex can effectively inhibit the occurrence of fibrosis of primary human trabecular meshwork cells, and in addition to glaucoma, it also has the potential to be developed into an inhibitor of other fibrotic diseases, such as pulmonary fibrosis, liver fibrosis, kidney fibrosis, myocardial fibrosis, etc.; and the preparation process of the mRNA-polypeptide complex is simple, which is conducive to large-scale production; it has high scientific research and technical application value and medical technology application prospect.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to an mRNA-polypeptide complex and a preparation method and application thereof. Background Art

[0002] Gene therapy refers to the introduction of exogenously produced nucleic acid substances into cells for the expression of corresponding proteins, which is used for disease-specific treatment or as a vaccine for disease prevention. As a new type of nucleic acid drug, messenger RNA (mRNA) has become a research hotspot in the fields of regenerative medicine, infectious disease vaccines, immunotherapy, etc. in recent years, and has great potential for clinical application. Compared with DNA-based gene therapy, mRNA therapy has many potential advantages: 1) mRNA does not need to be integrated into the cell genome, which can avoid the risk of insertional gene mutations and carcinogenesis; 2) The cell nucleus is the biggest barrier for nucleic acid drugs to enter cells and exert their effects, while mRNA can directly express functional proteins in the cytoplasm without crossing the barrier of the nuclear membrane, especially in non-dividing cells (such as neurons) or cells that divide very slowly (such as primary trabecular meshwork cells), which greatly improves the efficacy of nucleic acid drugs.

[0003] Glaucoma is a chronic neurodegenerative disease of the eye, characterized by optic disc cupping, progressive degeneration of retinal ganglion cells and optic nerve axons, accompanied by irreversible visual field loss and decreased vision, making it the world's leading irreversible blinding eye disease. Aqueous humor outflow obstruction caused by abnormal trabecular meshwork cell function is the main risk factor for increased intraocular pressure and the development of glaucoma. At present, drugs, surgery or lasers are mostly used clinically to control increased intraocular pressure, but there are defects such as significant side effects and low efficacy (drug treatment), as well as inevitable complications and limitations (surgery or laser treatment). A series of innovations from basic research to clinical transformation are urgently needed. Therefore, the development of new glaucoma treatment methods and strategies is of great medical value. Summary of the invention

[0004] The purpose of the present invention is to overcome the shortcomings and deficiencies of the prior art and to provide an mRNA-polypeptide complex and a preparation method and application thereof. The technical solution adopted by the present invention is as follows:

[0005] In the first aspect, an mRNA-polypeptide complex is provided, specifically including an mRNA containing a Smad7 gene sequence, namely, Smad7-mRNA, whose NCBI name is: Mus musculus Smad7 mRNA, complete cds, GenBank number is AF015260.1, base number: 1279 bp, and its corresponding cDNA specific sequence is shown in SEQ ID NO.1; the Smad7-mRNA is loaded on a polypeptide, and the polypeptide sequence is GHGVYGHGVYGHGPYGHGPYGHGLYW.

[0006] In a second aspect, a method for preparing the above-mentioned mRNA-polypeptide complex is provided, comprising the following steps:

[0007] S1. using mRNA in vitro synthesis technology to obtain mRNA containing the Smad7 gene sequence, namely Smad7-mRNA;

[0008] S2. A pure polypeptide powder containing 26 amino acids was prepared by solid phase synthesis, wherein the polypeptide sequence is GHGVYGHGVYGHGPYGHGPYGHGLYW;

[0009] S3. The polypeptide powder prepared in step S2 is dissolved in acetic acid to obtain a polypeptide solution, and the polypeptide solution is dropped into a PBS solution containing Smad7-mRNA to cause self-aggregation to obtain a Smad7-mRNA-polypeptide complex.

[0010] Preferably, in the preparation of the above mRNA-polypeptide complex, Smad7-mRNA with a concentration of 1 μg / mL and polypeptide with a concentration of 0.5 mg / mL are selected to prepare the Smad7-mRNA-polypeptide complex.

[0011] In a third aspect, the use of the above-mentioned mRNA-polypeptide complex in a pharmaceutical preparation is provided, specifically comprising:

[0012] Application of Smad7-mRNA-polypeptide complex in preparing drugs for inhibiting trabecular meshwork lesions, application of Smad7-mRNA-polypeptide complex in preparing drugs for delaying trabecular meshwork cell senescence, and application of Smad7-mRNA-polypeptide complex in preparing drugs for inhibiting trabecular meshwork cell fibrosis.

[0013] In summary, the present invention successfully obtains mRNA (Smad7-mRNA) containing the Smad7 gene sequence by using mRNA in vitro synthesis technology; prepares a pure polypeptide powder containing 26 amino acids by using a solid phase synthesis method; and prepares a Smad7-mRNA-polypeptide complex by a one-step method based on the principle of self-aggregation. The Smad7-mRNA-polypeptide complex can be used to inhibit trabecular meshwork lesions, and is expected to be developed into a new treatment strategy for glaucoma.

[0014] The beneficial effects of the present invention are as follows: ① The preparation process is simple, and only one-step method is required to prepare the mRNA-polypeptide complex, which is conducive to large-scale batch production; ② It has the function of significantly delaying the aging of primary human trabecular meshwork cells. In addition to glaucoma, it also has the potential to be developed into an inhibitor of other aging diseases, such as neurodegenerative diseases, skin aging, premature aging of stem cells, premature aging of immune cells, etc.; ③ It effectively inhibits the occurrence of fibrosis of primary human trabecular meshwork cells. In addition to glaucoma, it also has the potential to be developed into an inhibitor of other fibrosis diseases, such as pulmonary fibrosis, liver fibrosis, kidney fibrosis, myocardial fibrosis, etc.;

[0015] In summary, the Smad7-mRNA-polypeptide complex has high scientific research and technological application value and medical technology application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, without paying creative labor, other drawings obtained based on these drawings still belong to the scope of the present invention.

[0017] Figure 1 This is a map of the Smad7 in vitro transcription vector;

[0018] Figure 2 To identify the successful construction of Smad7 in vitro transcription vector using restriction enzyme digestion and agarose gel technology;

[0019] Figure 3 Agarose gel electrophoresis was used to identify the integrity and purity of Smad7-mRNA synthesized in vitro;

[0020] Figure 4 The senescence percentage of primary human trabecular meshwork cells in each group was observed and photographed using an optical microscope (A) and its representative images (B);

[0021] Figure 5 Western blotting was used to identify the fibrosis of primary human trabecular meshwork cells in each group. DETAILED DESCRIPTION

[0022] In order to make the objectives, technical solutions and advantages of the present invention more clear, the present invention will be further described in detail below in conjunction with embodiments and drawings.

[0023] Example

[0024] (1) Preparation of mRNA / peptide complex

[0025] Design and construct an in vitro transcription vector DNA containing the Smad7 sequence (pmRVac-mSmad7, Figure 1 ), enzyme digestion and agarose electrophoresis techniques proved that the plasmid DNA was successfully constructed ( Figure 2 The plasmid DNA obtained by amplification and purification of Escherichia coli was prepared into linear DNA, and Smad7-mRNA was synthesized by T7 based in vitro transcription, Cap1 capping, and PolyA tailing. Smad7-mRNA was purified by magnetic beads and analyzed by mRNA sequencing (100% alignment), gel electrophoresis ( Figure 3 ), UV spectroscopy (OD260 / 280: 2.12), and other methods confirmed that the synthesized Smad7-mRNA had good integrity, purity, and concentration.

[0026] The pure polypeptide powder was prepared by solid phase synthesis. The polypeptide contained 26 amino acids, and the specific sequence was: glycine-histidine-glycine-valine-tyrosine-glycine-histidine-glycine-valine-tyrosine-glycine-histidine-glycine-proline-tyrosine-glycine-histidine-glycine-proline-tyrosine-glycine-histidine-glycine-leucine-tyrosine-tryptophan (GHGVYGHGVYGHGPYGHGPYGHGLYW).

[0027] When the polypeptide solution (prepared in 10 mM acetic acid solution) was dropped into the Smad7-mRNA solution (prepared in PBS with a pH of 7.4), self-condensation occurred immediately to obtain the Smad7-mRNA-polypeptide complex.

[0028] (2) Function of mRNA-peptide complex in delaying cell aging

[0029] Through experimental comparison and screening, the inventors found that 0.5 mg / mL of polypeptide had the highest transfection efficiency, so 0.5 mg / mL of polypeptide was selected for the preparation of mRNA-polypeptide complexes;

[0030] Smad7-mRNA-peptide complex was prepared by using Smad7-mRNA with a concentration of 1 μg / mL and polypeptide with a concentration of 0.5 mg / mL. After the Smad7-mRNA-peptide complex was co-cultured with primary human trabecular meshwork cells for 4 hours, the Smad7-mRNA-peptide complex was removed, and the primary human trabecular meshwork cells were treated with TGF-β1 for 72 hours, and the cellular senescence was detected by using a β-galactosidase staining kit. The experimental results showed that the senescence rate of healthy primary human trabecular meshwork cells (control group) was 1.34±0.91%, and TGF-β1 treatment (TGF-β1 group) increased the senescence rate of primary human trabecular meshwork cells to 18.11±5.08%. The mRNA-peptide complex (TGF-β1+Smad7-mRNA-peptide complex group) could significantly reduce the senescence rate of primary human trabecular meshwork cells to 5.22±3.58%, indicating that the mRNA-peptide complex has the function of delaying the senescence of primary human trabecular meshwork cells ( Figure 4 ).

[0031] (3) Function of mRNA-peptide complex in inhibiting fibrosis

[0032] Smad7-mRNA with a concentration of 1 μg / mL and polypeptide with a concentration of 0.5 mg / mL were used to prepare Smad7-mRNA-polypeptide complexes; after co-culturing primary human trabecular meshwork cells with Smad7-mRNA-polypeptide complexes for 4 hours, the Smad7-mRNA-polypeptide complexes were removed, and primary human trabecular meshwork cells were treated with TGF-β1 for 24, 48 or 72 hours, total protein was extracted, and protein immunoblotting (Western blot) technology was used to detect changes in the expression of fibrosis marker proteins such as α-smooth muscle actin (α-SMA) and fibronectin. The experimental results showed that TGF-β1 treatment induced primary human trabecular meshwork cells to overexpress α-SMA and Fibronectin proteins, while mRNA-polypeptide complexes could inhibit the excessive abnormal expression of α-SMA and Fibronectin, indicating that mRNA-polypeptide complexes effectively inhibited the occurrence of fibrosis in primary human trabecular meshwork cells ( Figure 5 ).

[0033] The above shows that the mRNA-peptide complex has the function of effectively inhibiting trabecular meshwork lesions.

[0034] The above disclosure is only the preferred embodiment of the present invention, which certainly cannot be used to limit the scope of the present invention. Therefore, equivalent changes made according to the claims of the present invention are still within the scope of the present invention.

Claims

1. An mRNA-polypeptide complex, characterized in that: The invention comprises an mRNA containing a Smad7 gene sequence, namely Smad7-mRNA, whose GenBank number is AF015260.1; the Smad7-mRNA is loaded with a polypeptide, and the polypeptide sequence is GHGVYGHGVYGHGPYGHGPYGHGLYW.

2. The method for preparing an mRNA-polypeptide complex according to claim 1, characterized in that: The following steps are involved: S1. using mRNA in vitro synthesis technology to obtain mRNA containing the Smad7 gene sequence, namely Smad7-mRNA; S2. Preparing a polypeptide powder, the polypeptide sequence is GHGVYGHGVYGHGPYGHGPYGHGLYW; S3. The polypeptide powder prepared in step S2 is dissolved in acetic acid to obtain a polypeptide solution, and the polypeptide solution is dropped into a PBS solution containing Smad7-mRNA to cause self-aggregation to obtain a Smad7-mRNA-polypeptide complex.

3. The method for preparing an mRNA-polypeptide complex according to claim 2, characterized in that: The Smad7-mRNA-polypeptide complex was prepared by using Smad7-mRNA with a concentration of 1 μg / mL and polypeptide with a concentration of 0.5 mg / mL.

4. Use of the mRNA-polypeptide complex according to claim 1 in the preparation of a drug for delaying senescence of trabecular meshwork cells.

5. Use of the mRNA-polypeptide complex according to claim 1 in the preparation of a drug for inhibiting trabecular meshwork cell fibrosis.

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