Reprogramming factor preparation for promoting tissue cell proliferation and use

Abstract

A reprogramming factor preparation for promoting tissue cell proliferation and a use. The preparation comprises an mRNA encoding an alphavirus mutant replicase, an mRNA encoding an Oct4 transcription factor, an mRNA encoding a Sox2 transcription factor, an mRNA encoding a Klf4 transcription factor, an mRNA encoding a Glis1 transcription factor, and an mRNA encoding a Lin28 transcription factor. After the molar proportions of the mRNA expressing a replicase and the mRNAs expressing different transcription factors are adjusted and the above reprogramming factor preparation is introduced into the myocardium, synthesis of the transcription factors are achieved by means of the alphavirus mutant replicase, promoting cell division of cardiomyocytes and increase the number of cardiomyocytes, improving the cardiac function of an injured heart, especially improving the cardiac function after acute heart injuries; additionally, uncontrolled growth of cardiomyocytes is avoided, and hypertrophic cardiomyopathy and heart failure are avoided.

Description

Preparation and use of factors promoting tissue cell proliferation and reprogramming Technical Field

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

[0002] The treatment of heart failure is a major unmet clinical need. In the past 20 years, the disability and mortality rates of patients with heart failure have increased significantly. To date, there is a lack of therapies that can change the course of heart failure. Therefore, how to discover new and effective treatments is one of the most important missions of medicine in this century.

[0003] The loss of cardiomyocytes and / or loss of function is the pathological basis of heart failure. Coronary artery bypass grafting and orthotopic heart transplantation have provided new methods for the treatment of heart failure in recent years. However, cardiac fibrosis after treatment and severe shortage of donors have limited their clinical application.

[0004] The newly emerged cardiac cell transplantation method can supplement the damaged heart with new contractile cardiomyocytes, thereby improving the function of the damaged heart. However, cardiomyocytes are terminally differentiated cells, and adult cardiomyocytes have completely lost the ability to divide. Therefore, the clinical application of the above-mentioned cardiac cell transplantation method is greatly limited because it cannot provide enough contractile cardiomyocytes.

[0005] Furthermore, the field of cardiac gene therapy is progressing, but it has yet to achieve widespread clinical application. Currently, the most widely used gene therapy is delivered via viral vectors, particularly adeno-associated virus (AAV). Over the past few decades, adenovirus, adeno-associated virus (AAV), lentivirus, and DNA plasmids have been used to deliver genes of interest into cardiomyocytes. Both AAV and adenovirus achieve high myocardial transfection levels, while lentivirus and DNA plasmids have lower myocardial transfection efficiencies. However, adenovirus can provoke a strong immune response, making clinical application difficult, making AAV the only suitable option for cardiac gene therapy. However, the pharmacokinetics of AAV in the heart (expression begins on day 4 and persists for at least 11 months) can lead to uncontrolled myocardial growth, hypertrophic cardiomyopathy, and heart failure. Furthermore, over 60% of healthy humans have neutralizing antibodies against the AAV capsid, which effectively neutralize gene expression delivered via this method. These drawbacks limit the application of AAV in cardiac gene therapy. Furthermore, the potential for severe immune reactions (allergic reactions) to AAV when delivering target proteins via AAV is compounded.

[0006] Currently, there is an urgent need to propose a new cardiac gene therapy drug without the risk of genomic integration.

[0007] Summary of the Invention

[0008] In view of the above problems, the embodiments of the present application provide a circuit, a chip and an electronic device to solve the above technical problems.

[0009] In a first aspect, an embodiment of the present application provides a tissue cell proliferation reprogramming factor preparation, comprising 7 to 8 molar parts of an mRNA nucleic acid molecule encoding a mutant alphavirus replicase, 2 to 4 molar parts of an mRNA nucleic acid molecule encoding an Oct4 transcription factor, 1 molar part of an mRNA nucleic acid molecule encoding a Sox2 transcription factor, 1 molar part of an mRNA nucleic acid molecule encoding a Klf4 transcription factor, 1 molar part of an mRNA nucleic acid molecule encoding a Glis1 transcription factor, and 1 molar part of an mRNA nucleic acid molecule encoding a Lin28 transcription factor.

[0010] Optionally, it includes 7 to 8 molar parts of an mRNA nucleic acid molecule encoding an alphavirus mutant replicase, 3 molar parts of an mRNA nucleic acid molecule encoding an Oct4 transcription factor, 1 molar part of an mRNA nucleic acid molecule encoding a Sox2 transcription factor, 1 molar part of an mRNA nucleic acid molecule encoding a Klf4 transcription factor, 1 molar part of an mRNA nucleic acid molecule encoding a Glis1 transcription factor, and 1 molar part of an mRNA nucleic acid molecule encoding a Lin28 transcription factor.

[0011] Optionally, the tissue cell proliferation-promoting reprogramming factor preparation further includes a first solvent, the concentration of citrate in the first solvent is 0-15 mmol / L, and the concentration of sodium chloride in the first solvent is 120-140 mmol / L.

[0012] Optionally, the concentration of citrate in the first solvent is 10 mmol / L, the concentration of sodium chloride is 130 mmol / L, and the concentration of sucrose is 0-0.1 g / mL, and the pH value of the first solvent is 7.5;

[0013] Alternatively, the concentration of citrate in the first solvent is 10 mmol / L, the concentration of sodium chloride is 130 mmol / L, and the concentration of sodium dihydrogen phosphate is 0-2 mmol / L, and the pH value of the first solvent is 7.5.

[0014] 5. The tissue cell proliferation-promoting reprogramming factor preparation according to claim 1, characterized in that each of the mRNA nucleic acid molecules comprises a 5' cap structure, a 5' UTR sequence, a coding sequence of the corresponding transcription factor mRNA or a coding sequence of the alphavirus mutant replicase mRNA, a 3' UTR sequence and a polyadenylation sequence.

[0015] Optionally, some or all of the nucleotides in the mRNA nucleic acid molecule encoding the mutant alphavirus replicase, the mRNA nucleic acid molecule encoding the Oct4 transcription factor, the mRNA nucleic acid molecule encoding the Sox2 transcription factor, the mRNA nucleic acid molecule encoding the Klf4 transcription factor, the mRNA nucleic acid molecule encoding the Glis1 transcription factor, and the mRNA nucleic acid molecule encoding the Lin28 transcription factor have been chemically modified to improve the stability of the mRNA nucleic acid molecules in the organism.

[0016] Optionally, the chemical modification includes replacing 100% of the cytosine in the mRNA nucleic acid molecule encoding the mutant alphavirus replicase, the mRNA nucleic acid molecule encoding the Oct4 transcription factor, the mRNA nucleic acid molecule encoding the Sox2 transcription factor, the mRNA nucleic acid molecule encoding the Klf4 transcription factor, the mRNA nucleic acid molecule encoding the Glis1 transcription factor, and the mRNA nucleic acid molecule encoding the Lin28 transcription factor with 5-methylcytosine, and replacing 100% of the uracil in the mRNA nucleic acid molecule encoding the mutant alphavirus replicase, the mRNA nucleic acid molecule encoding the Oct4 transcription factor, the mRNA nucleic acid molecule encoding the Sox2 transcription factor, the mRNA nucleic acid molecule encoding the Klf4 transcription factor, the mRNA nucleic acid molecule encoding the Glis1 transcription factor, and the mRNA nucleic acid molecule encoding the Lin28 transcription factor with N1-methylpseudouridine.

[0017] Optionally, the mutant replicase produces a mutation at position 259 of the nsP2 region and a mutation at position 650 of the nsP2 region, wherein the mutation at position 259 of the nsP2 region is from serine S to proline P, and the mutation at position 650 of the nsP2 region is from arginine R to aspartic acid D.

[0018] In a second aspect, the embodiments of the present application provide a use of the above-mentioned tissue cell proliferation-promoting reprogramming factor preparation in promoting cardiomyocyte proliferation or in the preparation of cardiac gene therapy drugs.

[0019] In a third aspect, the present invention provides an embodiment of the present invention for use of the above-mentioned tissue cell proliferation-promoting reprogramming factor preparation in promoting chondrocyte proliferation or in preparing a drug for bone and joint repair.

[0020] The present invention provides a tissue cell proliferation-promoting reprogramming factor preparation and its use, comprising 7 to 8 molar parts of an mRNA nucleic acid molecule encoding a mutant alphavirus replicase, 2 to 4 molar parts of an mRNA nucleic acid molecule encoding an Oct4 transcription factor, 1 molar part of an mRNA nucleic acid molecule encoding a Sox2 transcription factor, 1 molar part of an mRNA nucleic acid molecule encoding a Klf4 transcription factor, 1 molar part of an mRNA nucleic acid molecule encoding a Glis1 transcription factor, and 1 molar part of an mRNA nucleic acid molecule encoding a Lin28 transcription factor. By adjusting the molar ratio of the mRNA expressing the replicase and the mRNA nucleic acid molecules of different transcription factors in the above manner, a specific tissue cell proliferation-promoting reprogramming factor preparation is formed. After the above reprogramming factor preparation is introduced into the myocardium, the mutant alphavirus replicase is synthesized. The synthesis of the above transcription factors is achieved by the mutant alphavirus replicase, which can promote myocardial cell division and increase the number of myocardial cells, thereby improving the cardiac function of the damaged heart, especially improving the cardiac function of acute heart injury. At the same time, the uncontrolled growth of myocardial cells is avoided, and the occurrence of hypertrophic cardiomyopathy and heart failure is avoided.

[0021] These and other aspects of the present application will become more readily apparent from the description of the following embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] FIG1 shows a comparison chart of the change in fluorescein intensity with injection time in Example 3 of the present application.

[0023] FIG2 shows a bar graph comparing the fluorescein intensity of Example 3 of the present application.

[0024] FIG3 shows a comparison diagram of the experimental site of Example 4 of the present application.

[0025] FIG4 shows a comparison chart of the proportions of mice with myocardial infarction that tolerate forced electric shock treadmill according to Example 4 of the present application.

[0026] FIG5 shows a comparison chart of the forced electric shock tolerance treadmill time in Example 4 of the present application.

[0027] FIG6 shows a comparison chart of forced electric shock treadmill distances in Example 4 of the present application.

[0028] FIG7 shows a comparison diagram of scar areas after myocardial infarction in Example 4 of the present application.

[0029] FIG8 shows a comparison diagram of cardiomyocyte proliferation in Example 4 of the present application.

[0030] FIG9 shows a comparison diagram of the myocardial protection effect of Example 4 of the present application.

[0031] FIG10 shows a comparison of expressions of different experimental groups in Examples 5 and 6 of the present application.

[0032] FIG11 shows a comparison of the expression changes in the bone joint cavity of different experimental groups in Example 5 and Example 6 of the present application.

[0033] FIG12 shows a comparison of IFN-a changes in different experimental groups in Examples 5 and 6 of the present application.

[0034] FIG13 shows a microscopic comparison diagram of cartilage thickness in Example 7 of the present application.

[0035] FIG14 shows a comparison chart of the cartilage thickness detection results of Example 7 of the present application.

[0036] FIG15 shows the results of evaluating the therapeutic efficacy of different ratios of reprogramming factor combinations on myocardial cells according to Example 8 of the present application. DETAILED DESCRIPTION

[0037] The embodiments of the present application are described in detail below. Examples of the embodiments are shown in the accompanying drawings, wherein the same or similar reference numerals throughout represent the same or similar elements or elements having the same or similar functions. The embodiments described below with reference to the accompanying drawings are exemplary and are only used to explain the present application and are not to be construed as limiting the present application.

[0038] In order to enable those skilled in the art to better understand the solutions of the present application, the technical solutions in the embodiments of the present application will be clearly and completely described below in conjunction with the drawings in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, not all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without making creative efforts are within the scope of protection of this application.

[0039] In the embodiments of the present application, it should be noted that, in this document, relational terms such as first and second, etc., are merely used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any actual relationship or order between these entities or operations.

[0040] Moreover, the terms "comprises," "comprising," or any other variations thereof are intended to encompass non-exclusive inclusion, such that a process, method, article, or apparatus that includes a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, method, article, or apparatus. In the absence of further limitations, an element defined by the phrase "comprising a ..." does not preclude the presence of additional identical elements in the process, method, article, or apparatus that includes the element.

[0041] In the description of the embodiments of this application, words such as "example" or "for example" are used to indicate an example, illustration, or description. Any embodiment or design described as "for example" or "for example" in the embodiments of this application is not to be construed as being preferred or having more advantages than another embodiment or design. The use of words such as "example" or "for example" is intended to clearly present relative concepts.

[0042] In addition, in the embodiments of the present application, "plurality" refers to two or more. In view of this, in the embodiments of the present application, "plurality" can also be understood as "at least two". "At least one" can be understood as one or more, for example, one, two, or more. For example, "including at least one" means including one, two, or more, and does not limit which ones are included. For example, "including at least one of A, B, and C" means including A, B, C, A and B, A and C, B and C, or A, B, and C.

[0043] It should be noted that in the embodiments of the present application, "and / or" describes the association relationship between associated objects, indicating that three relationships can exist. For example, A and / or B can represent three situations: A exists alone, A and B exist simultaneously, and B exists alone. In addition, the character " / ", unless otherwise specified, generally indicates that the associated objects are in an "or" relationship.

[0044] The experimental methods in the following examples are conventional methods unless otherwise specified.

[0045] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0046] One embodiment of the present application provides a tissue cell proliferation-promoting reprogramming factor preparation, comprising 7 to 8 molar parts of an mRNA nucleic acid molecule encoding an alphavirus mutant replicase, 2 to 4 molar parts of an mRNA nucleic acid molecule encoding an Oct4 transcription factor, 1 molar part of an mRNA nucleic acid molecule encoding a Sox2 transcription factor, 1 molar part of an mRNA nucleic acid molecule encoding a Klf4 transcription factor, 1 molar part of an mRNA nucleic acid molecule encoding a Glis1 transcription factor, and 1 molar part of an mRNA nucleic acid molecule encoding a Lin28 transcription factor.

[0047] In this embodiment, a specific tissue cell proliferation-promoting reprogramming factor preparation is formed by adjusting the molar ratio of mRNA nucleic acid molecules expressing different transcription factors. After the above-mentioned reprogramming factor preparation is introduced into the myocardium, a mutant alphavirus replicase is synthesized. The synthesis of the above-mentioned transcription factors is achieved by the mutant alphavirus replicase, which can promote myocardial cells to enter cell division and increase the number of myocardial cells, thereby improving the cardiac function of the damaged heart, especially improving the cardiac function of acute heart injury; at the same time, it avoids the uncontrolled growth of myocardial cells and avoids the occurrence of hypertrophic cardiomyopathy and heart failure.

[0048] As an embodiment, the tissue cell proliferation reprogramming factor preparation includes 7 to 8 molar parts of an mRNA nucleic acid molecule encoding an alphavirus mutant replicase, 3 molar parts of an mRNA nucleic acid molecule encoding an Oct4 transcription factor, 1 molar part of an mRNA nucleic acid molecule encoding a Sox2 transcription factor, 1 molar part of an mRNA nucleic acid molecule encoding a Klf4 transcription factor, 1 molar part of an mRNA nucleic acid molecule encoding a Glis1 transcription factor, and 1 molar part of an mRNA nucleic acid molecule encoding a Lin28 transcription factor.

[0049] In one embodiment, the tissue cell proliferation reprogramming factor preparation further includes a first solvent, wherein the concentration of citrate in the first solvent is 0 to 15 mmol / L, and the concentration of sodium chloride in the first solvent is 120 to 140 mmol / L. The aforementioned mRNA nucleic acid molecule encoding the mutant alphavirus replicase, the mRNA nucleic acid molecule encoding the Oct4 transcription factor, the mRNA nucleic acid molecule encoding the Sox2 transcription factor, the mRNA nucleic acid molecule encoding the Klf4 transcription factor, the mRNA nucleic acid molecule encoding the Glis1 transcription factor, and the mRNA nucleic acid molecule encoding the Lin28 transcription factor are dissolved in the first solvent, which can be formed by a mixture of citrate and physiological saline.

[0050] In this embodiment, the first solvent can make each mRNA nucleic acid molecule less susceptible to degradation.

[0051] In some embodiments, the concentration of citrate in the first solvent is 10 mmol / L, the concentration of sodium chloride is 130 mmol / L, and the concentration of sucrose is 0-0.1 g / mL, and the pH value of the first solvent is 7.5;

[0052] In some embodiments, the concentration of citrate in the first solvent is 10 mmol / L, the concentration of sodium chloride is 130 mmol / L, and the concentration of sodium dihydrogen phosphate is 0-2 mmol / L, and the pH value of the first solvent is 7.5.

[0053] In some embodiments, the concentration of citrate in the first solvent is 10 mmol / L, the concentration of sodium chloride is 130 mmol / L, the concentration of sodium dihydrogen phosphate is 0-2 mmol / L, and the concentration of sucrose is 0-0.1 g / mL, and the pH value of the first solvent is 7.5.

[0054] As an embodiment, each mRNA nucleic acid molecule includes a 5' cap structure, a 5' UTR sequence, a coding sequence of a corresponding transcription factor mRNA or a coding sequence of an alphavirus mutant replicase mRNA, a 3' UTR sequence and a polyadenylation sequence.

[0055] Specifically, the mRNA nucleic acid molecule encoding the mutant replicase of the genus alphavirus includes a 5' cap structure, a 5' UTR sequence, an mRNA coding sequence of the mutant replicase of the genus alphavirus, a 3' UTR sequence and a polyadenylic acid sequence; the mRNA nucleic acid molecule encoding the Oct4 transcription factor includes a 5' cap structure, a 5' UTR sequence, an mRNA coding sequence of the Oct4 transcription factor, a 3' UTR sequence and a polyadenylic acid sequence; the mRNA nucleic acid molecule encoding the Sox2 transcription factor includes a 5' cap structure, a 5' UTR sequence, an mRNA coding sequence of the Sox2 transcription factor, a 3' UTR sequence and a polyadenylic acid sequence. column; the mRNA nucleic acid molecule encoding the Klf4 transcription factor includes a 5' cap structure, a 5' UTR sequence, the mRNA coding sequence of the Klf4 transcription factor, a 3' UTR sequence and a polyadenylic acid sequence; the mRNA nucleic acid molecule encoding the Glis1 transcription factor includes a 5' cap structure, a 5' UTR sequence, the mRNA coding sequence of the Glis1 transcription factor, a 3' UTR sequence and a polyadenylic acid sequence; the mRNA nucleic acid molecule encoding the Lin28 transcription factor includes a 5' cap structure, a 5' UTR sequence, the mRNA coding sequence of the Lin28 transcription factor, a 3' UTR sequence and a polyadenylic acid sequence.

[0056] As an embodiment, some or all of the nucleotides in the mRNA nucleic acid molecule encoding the mutant alphavirus replicase, the mRNA nucleic acid molecule encoding the Oct4 transcription factor, the mRNA nucleic acid molecule encoding the Sox2 transcription factor, the mRNA nucleic acid molecule encoding the Klf4 transcription factor, the mRNA nucleic acid molecule encoding the Glis1 transcription factor, and the mRNA nucleic acid molecule encoding the Lin28 transcription factor are chemically modified to improve the stability of the mRNA nucleic acid molecules in vivo.

[0057] In some embodiments, the chemical modification includes replacing 100% of the cytosine in the mRNA nucleic acid molecule encoding the mutant alphavirus replicase, the mRNA nucleic acid molecule encoding the Oct4 transcription factor, the mRNA nucleic acid molecule encoding the Sox2 transcription factor, the mRNA nucleic acid molecule encoding the Klf4 transcription factor, the mRNA nucleic acid molecule encoding the Glis1 transcription factor, and the mRNA nucleic acid molecule encoding the Lin28 transcription factor with 5-methylcytosine, and replacing 100% of the uracil in the mRNA nucleic acid molecule encoding the mutant alphavirus replicase, the mRNA nucleic acid molecule encoding the Oct4 transcription factor, the mRNA nucleic acid molecule encoding the Sox2 transcription factor, the mRNA nucleic acid molecule encoding the Klf4 transcription factor, the mRNA nucleic acid molecule encoding the Glis1 transcription factor, and the mRNA nucleic acid molecule encoding the Lin28 transcription factor with N1-methylpseudouridine.

[0058] As an embodiment, the mutant replicase produces a mutation at position 259 of the nsP2 region and a mutation at position 650 of the nsP2 region, wherein the mutation at position 259 of the nsP2 region is from serine S to proline P, and the mutation at position 650 of the nsP2 region is from arginine R to aspartic acid D.

[0059] In this embodiment, after the above-mentioned reprogramming factor preparation is introduced into the myocardium, a mutant alphavirus replicase is synthesized. The synthesis of the above-mentioned transcription factors is achieved through the mutant alphavirus replicase, which can achieve limited self-replication of each transcription factor, avoid the uncontrolled growth of myocardial cells, avoid the occurrence of hypertrophic cardiomyopathy and heart failure, and improve the safety of the reprogramming factor preparation.

[0060] In other embodiments, the reprogramming factor preparation may not include the mRNA nucleic acid molecule encoding the mutant alphavirus replicase, and the replicase in cardiomyocytes may be used to synthesize the transcription factors.

[0061] One embodiment of the present application further provides the use of the above-mentioned tissue cell proliferation-promoting reprogramming factor preparation in promoting cardiomyocyte proliferation or in the preparation of cardiac gene therapy drugs.

[0062] Among them, the reprogramming factor preparation is introduced into the myocardial muscle by injection, and the reprogramming factors are produced and expressed by the body's own heart cells, thereby initiating partial epigenetic reprogramming. The epigenetic expression of myocardial cells is readjusted, and the epigenetic state of myocardial cells is reversed to be closer to a younger state, thereby quickly promoting the repair and functional improvement of the damaged heart.

[0063] One embodiment of the present application further provides the use of the above-mentioned tissue cell proliferation-promoting reprogramming factor preparation in promoting chondrocyte proliferation or in the preparation of bone and joint repair drugs.

[0064] Among them, the inventors of the present application unexpectedly discovered that the reprogramming factor preparation can be efficiently expressed in the bone joint cavity, skin and muscle, and can restore the regeneration of damaged cartilage.

[0065] Example 1: Preparation of a tissue cell proliferation-promoting reprogramming factor preparation

[0066] A) Preparation of mRNA encoding mutant alphavirus replicase

[0067] Step 1: Using GeneArtTM Gibson HiFi reaction (Thermo Fisher, USA, A46624) was used to synthesize the mutant replicase DNA coding sequence (a DNA nucleic acid molecule encoding the mutant replicase of the alphavirus genus and without a polyadenylation sequence). After successful synthesis, the mutant replicase DNA coding sequence was cloned into the pcDNA3.3 vector plasmid for industrial production.

[0068] 1.1 Mutant replicase DNA coding sequence: 5' untranslated region DNA sequence (SEQ ID NO. 9), mutant replicase coding sequence, and 3' untranslated region DNA sequence (SEQ ID NO. 10). The mutant replicase coding sequence is divided into four DNA fragments: the nsP1 region fragment (SEQ ID NO. 3), the nsP2 region fragment (SEQ ID NO. 4), the nsP3 region fragment (SEQ ID NO. 5), and the nsP4 region fragment (SEQ ID NO. 6). All four DNA fragments are modified fragments with high GC content. The four DNA fragments were ordered directly from IDT Corporation in the United States as gblocks.

[0069] Specifically, the steps include: assembling a Gibson reaction according to Table 1, reacting at 50° C. for 60 minutes in a PCR instrument, and obtaining a PCR product.

[0070] Table 1 Gibson reaction system

[0071] 1.2 Transform the PCR product into One Shot™ TOP10 chemically competent E. coli cells, including the following steps:

[0072] Dilute the Gibson reaction system (PCR product) with nuclease-free water at a ratio of 1:5, add 12 μL of nuclease-free water and 3 μL of Gibson reaction system, mix well, and react on ice;

[0073] Add 1 μL of the dilution to One Shot™ TOP10 chemically competent E. coli cells and mix. Incubate the transformation mixture on ice for 20–30 minutes.

[0074] Incubate the cells at 42°C for 30 seconds without shaking;

[0075] Immediately transfer the reaction tube to ice and incubate on ice for 2 minutes;

[0076] 450 μL of room temperature SOC culture medium (Life Technology, USA) was added;

[0077] Shake at 300 rpm at 37°C for 1 hour;

[0078] Take 100 μL and apply it to a bacterial culture plate (100 μg / mL ampicillin or 50 μg / mL kanamycin.);

[0079] The cells were incubated at 37°C overnight, bacterial clones were selected, and the cells were shaken at 37°C. The double-mutant replicase sequence plasmid containing the correct sequence was selected by first-generation sequencing.

[0080] Step 2: Add the poly-(a) tail of mRNA by PCR to obtain the DNA synthesis template of replicase mRNA

[0081] The poly-(a) tail consists of 120 adenosine nucleotides.

[0082] Prepare PCR master mix according to Table 2 (total volume 200 μL, 25 μL for each of eight reactions);

[0083] Table 2 Composition of PCR premix

[0084] PCR reaction was performed according to the reaction conditions shown in Table 3;

[0085] Table 3 PCR reaction conditions

[0086] Check the quality of PCR products by gel electrophoresis;

[0087] The PCR product was recovered by gel excision (QIAquick PCR purification kit, Qiagen, cat. no. 28106), and the final concentration of the tail template was adjusted to 100 ng / μL, which was used as a template for DNA synthesis of replicase mRNA encoding in vitro transcription.

[0088] Step 3: In vitro transcription synthesis of replicase encoding mRNA

[0089] 1. Assemble the mRNA cap structure and nucleotide mixture according to Table 4:

[0090] The cap structure was 3′-O-Me-m7G(5′)ppp(5′)G RNA cap analog (New England Biolabs, cat. no. S1411S), -Methylcytidine-5′-triphosphate (Me-CTP; Trilink, cat. no. N1014), N1-methyl-pseudo-UTP (Trilink, cat. no. N1019), and other components were from the MEGAscript T7 kit (Ambion, cat. no. AM1334).

[0091] Table 4 mRNA cap structure and nucleotide mixture

[0092] 2. Assemble the in vitro transcription system encoding replicase mRNA according to Table 5:

[0093] Table 5 In vitro transcription system of replicase encoding mRNA

[0094] 3. Place the reaction in a PCR instrument and incubate at 37°C for 3 to 6 hours.

[0095] 4. Add 2 μL of Turbo DNase (from MEGAscript T7 kit, Ambion, cat. no. AM1334) to each sample.

[0096] 5. Mix gently and incubate at 37°C for 15 minutes.

[0097] 6. Purify the reaction treated with DNase and RNAseIII using the MEGAclear kit (Ambion, cat. no. AM1908); elute the modified mRNA with a total of 100 μL of elution buffer (elute twice with 50 μL of elution buffer).

[0098] 7. Treat the purified modified mRNA with Antarctic phosphatase (New England Biolabs, cat. no. M0289S).

[0099] 8. To each sample (~100 μL), add 11 μL of 10× phosphatase buffer, followed by 2 μL of phosphatase; gently mix the samples and incubate at 37°C for 0.5-1 h.

[0100] 9. After elution, measure the concentration of the modified replicase-encoding mRNA in a NanoDrop spectrophotometer. The expected total yield should be ~50 μg (30-70 μg range; a 100 μL elution volume from a 40 μL IVT reaction is 300-700 ng / μL). Adjust the concentration to 100 ng / μL by adding elution buffer or TE buffer (pH 7.0), or purify by FPLC.

[0101] B) Preparation of mRNA encoding each transcription factor

[0102] The synthesis steps of mRNA encoding transcription factors are similar to those of mRNA encoding replicase, including the following steps:

[0103] Step 1: Using GeneArtTM Gibson HiFi reaction (Thermo Fisher, USA, A46624) was used to synthesize the specifically modified target factor DNA coding sequence (the nucleic acid molecule encoding the transcription factor mRNA does not contain a polyadenylation sequence);

[0104] Among them, the specifically modified target factor DNA coding sequence: 5' untranslated region DNA sequence (SEQ ID NO.9), replicase 5' end specific DNA sequence (SEQ ID NO.7), target factor DNA coding sequence (see Table 6), replicase 3' end specific DNA sequence (SEQ ID NO.8), 3' untranslated region DNA sequence (SEQ ID NO.10).

[0105] Step 2: Adding the poly-(a) tail of mRNA to the specifically modified target factor DNA coding sequence by PCR to obtain a DNA synthesis template encoding the transcription factor mRNA;

[0106] Step 3: In vitro transcription to synthesize mRNA encoding transcription factors.

[0107] The five transcription factor encoding mRNAs shown in Table 6 were synthesized according to the above method.

[0108] Table 6 DNA synthesis templates for different mRNA encoding transcription factors

[0109] C) The molar ratio of mRNA encoding the mutant alphavirus replicase, mRNA encoding the Oct4 transcription factor, mRNA encoding the Sox2 transcription factor, mRNA encoding the Klf4 transcription factor, mRNA encoding the Glis1 transcription factor, and mRNA encoding the Lin28 transcription factor is 7:3:1:1:1:1, and they are dissolved in the first solvent (10 mmol / L citrate, 130 mmol / L sodium chloride, 0.15 g / mL sucrose in nuclease-free distilled water, pH 7.5).

[0110] Example 2: Preparation of different mRNA encoding fluorescent proteins

[0111] The synthesis steps of mRNA encoding fluorescent protein are similar to those of mRNA encoding replicase, including the following steps:

[0112] Step 1: Using GeneArtTM Gibson HiFi reaction (Thermo Fisher, USA, A46624) was used to synthesize the specifically modified fluorescent protein DNA coding sequence (the nucleic acid molecule encoding the fluorescent protein mRNA does not contain a polyadenylation sequence);

[0113] Among them, the specifically modified fluorescent protein DNA coding sequence: 5' untranslated region DNA sequence (SEQ ID NO.9), replicase 5' end specific DNA sequence (SEQ ID NO.7), target fluorescent protein DNA coding sequence (see Table 7), replicase 3' end specific DNA sequence (SEQ ID NO.8), 3' untranslated region DNA sequence (SEQ ID NO.10).

[0114] Step 2: Adding the mRNA poly-(a) tail to the specifically modified target fluorescent protein DNA coding sequence by PCR to obtain a DNA synthesis template encoding fluorescent protein mRNA;

[0115] Step 3: In vitro transcription to synthesize mRNA encoding fluorescent protein.

[0116] According to the above method, the two mRNAs encoding transcription factors shown in Table 7 were synthesized respectively.

[0117] Table 7 DNA synthesis templates for different mRNAs encoding fluorescent proteins

[0118] Example 2 - eGFP protein mRNA preparation: mRNA encoding the mutant replicase of the alphavirus and mRNA encoding the eGFP protein were dissolved in a first solvent (10 mmol / L citrate, 130 mmol / L sodium chloride, 0.15 g / mL sucrose, nuclease-free distilled water, pH 7.5).

[0119] Example 2 - Firefly luciferin protein mRNA preparation: mRNA encoding the mutant replicase of the alphavirus genus and mRNA encoding the firefly luciferin protein are dissolved in a first solvent (10 mmol / L citrate, 130 mmol / L sodium chloride, 0.15 g / mL sucrose nuclease-free distilled water, pH 7.5).

[0120] Comparative Example 1: Ordinary firefly luciferin protein mRNA preparation

[0121] The mRNA encoding the eGFP protein was dissolved in a first solvent (10 mmol / L citrate, 130 mmol / L sodium chloride, 0.15 g / mL sucrose, nuclease-free distilled water, pH 7.5).

[0122] Comparative Example 2: Empty mRNA Preparation

[0123] The first solvent (10 mmol / L citrate, 130 mmol / L sodium chloride, 0.15 g / mL sucrose, nuclease-free distilled water, pH 7.5).

[0124] Comparative Example 3: mRNA encoding the mutant alphavirus replicase and mRNA encoding the firefly luciferin protein were dissolved in physiological saline.

[0125] Comparative Example 4: mRNA encoding a mutant alphavirus replicase and mRNA encoding a firefly luciferin protein were dissolved in a second solvent (10 mmol / L citrate, 130 mmol / L sodium chloride, pH 7.5).

[0126] Comparative Example 5:

[0127] mRNA encoding a mutant alphavirus replicase and mRNA encoding a firefly luciferin protein, dissolved in a calcium phosphate solution.

[0128] Comparative Example 6:

[0129] mRNA encoding the mutant alphavirus replicase and mRNA encoding the firefly luciferin protein were dissolved in RNAimax transfection reagent (Life Technologies).

[0130] Comparative Example 7:

[0131] mRNA encoding the mutant alphavirus replicase and mRNA encoding the firefly luciferin protein were dissolved in Invivo JetPEI transfection reagent (Polyplus).

[0132] Comparative Example 8:

[0133] mRNA encoding the mutant alphavirus replicase and mRNA encoding the firefly luciferin protein were dissolved in MC3 nanoliposomes (Cayman Chem).

[0134] Comparative Example 9:

[0135] The synthesis steps of mRNA encoding vegfa protein are similar to those of mRNA encoding replicase, including the following steps:

[0136] Step 1: Using GeneArtTM Gibson The specifically modified vegfa protein DNA coding sequence (the nucleic acid molecule encoding the vegfa protein mRNA does not contain a polyadenylation sequence) was synthesized by HiFi reaction (Thermo Fisher, USA, A46624);

[0137] Among them, the specifically modified vegfa protein DNA coding sequence: 5' untranslated region DNA sequence (SEQ ID NO.9), replicase 5' end specific DNA sequence (SEQ ID NO.7), vegfa protein DNA coding sequence, replicase 3' end specific DNA sequence (SEQ ID NO.8), 3' untranslated region DNA sequence (SEQ ID NO.10).

[0138] Step 2: Adding the poly-(a) tail of mRNA to the specifically modified vegfa protein DNA coding sequence by PCR to obtain a DNA synthesis template encoding vegfa protein mRNA;

[0139] Step 3: In vitro transcription to synthesize mRNA encoding vegfa protein.

[0140] The preparation includes mRNA encoding mutant alphavirus replicase and mRNA encoding vegfa protein, which are dissolved in a first solvent (10 mmol / L citrate, 130 mmol / L sodium chloride, 0.15 g / mL sucrose, nuclease-free distilled water, pH 7.5).

[0141] Comparative Example 10:

[0142] The molar ratio of mRNA encoding the mutant replicase of the alphavirus, mRNA encoding the Oct4 transcription factor, mRNA encoding the Sox2 transcription factor, mRNA encoding the Klf4 transcription factor, mRNA encoding the Glis1 transcription factor, and mRNA encoding the Lin28 transcription factor is 7:1:1:1:1:1, and they are dissolved in the first solvent (10 mmol / L citrate, 130 mmol / L sodium chloride, 0.15 g / mL sucrose in nuclease-free distilled water, pH 7.5).

[0143] Comparative Example 11:

[0144] The molar ratio of mRNA encoding the mutant replicase of the alphavirus genus, mRNA encoding the Oct4 transcription factor, mRNA encoding the Sox2 transcription factor, mRNA encoding the Klf4 transcription factor, mRNA encoding the Glis1 transcription factor, and mRNA encoding the Lin28 transcription factor is 7:0.5:1:1:1:1, and they are dissolved in the first solvent (10 mmol / L citrate, 130 mmol / L sodium chloride, 0.15 g / mL sucrose in nuclease-free distilled water, pH 7.5).

[0145] Comparative Example 12:

[0146] mRNA encoding the mutant alphavirus replicase and mRNA encoding the eGFP protein were dissolved in MC3 nanoliposomes (Cayman Chem).

[0147] Example 3: Expression imaging experiment

[0148] The expression of the mRNA preparation of the mouse heart-specific reporter gene green fluorescent protein (eGFP) or firefly luciferin protein was verified by intramyocardial injection. The expression of the firefly fluorescent protein (Luci) mRNA preparation was quantitatively detected by an IVIS imager. The specific steps are as follows: the left chest area of ​​the mouse was exposed, the chest cavity was incised, and the heart was exposed. The mouse was divided into 9 experimental groups. The 9 experimental groups were injected with the reporter gene modified mRNA preparation of Example 2 above, the ordinary firefly luciferin protein mRNA preparation of Comparative Example 1, the empty mRNA preparation of Comparative Example 2, the preparation of Comparative Example 3, the preparation of Comparative Example 4, the mRNA nanoliposome particle RNAimax delivery preparation of Comparative Example 5, the RNAimax delivery preparation of Comparative Example 6, the Invivo JetPEI delivery preparation of Comparative Example 7, and the MC3 nanoliposome of Comparative Example 8. Comparative Examples 5 to 8 were respectively delivered by limited self-replicating mRNA intramyocardial injection according to the manufacturer's instructions.

[0149] Experimental results and analysis:

[0150] Please refer to Figures 1 and 2. The firefly luciferin protein mRNA preparation of Example 2 of the present application is efficiently expressed in the myocardium. As shown in Figure 1, the peak expression time of the single limited self-replicating firefly luciferin protein mRNA preparation of Example 2 is 480 hours after injection, and the duration of firefly luciferin protein expression is 1300 hours after injection. Compared with the ordinary firefly luciferin mRNA preparation of Comparative Example 1, the peak expression and duration of Example 2 are significantly improved.

[0151] As shown in Figure 2, the formulation of Example 2 has the highest expression of reporter gene limited self-replication mRNA compared with other formulation components (Comparative Examples 3 to Comparative Examples 8). In cardiac muscle injection applications, the expression of reporter gene limited self-replication mRNA delivered by non-liposomal delivery is significantly higher than that of reporter gene mRNA delivered by liposomes.

[0152] Please refer to Figure 12. After the preparation of Example 2 was injected into the myocardium, compared with the preparation of Comparative Example 8, the IFN-a level in the mouse serum was not significantly increased 24 hours after injection of the preparation of Example 2, indicating that the mRNA preparation of Example 2 had no immune response compared to the MC3 nanoparticles.

[0153] Example 4: Myocardial Therapeutic Effect Evaluation Experiment

[0154] The myocardial infarction model was established by ligating the left anterior descending coronary artery in mice and the preparation of Example 1 was used for treatment and efficacy evaluation. The specific steps are as follows:

[0155] 1. Establishment of a myocardial infarction model by ligating the left anterior descending branch of the coronary artery in mice: Select suitable C57 mice and anesthetize them with isoflurane. Before the operation, disinfect the chest surface of the mouse. Make a midline incision in the mouse chest and carefully expose the heart with surgical scissors or a dissector. Use microsurgery techniques and a magnifying glass to find the left anterior descending artery of the mouse heart and ligate it with an absorbable suture. The ligation position is located near the heart of the coronary artery, which will cause myocardial ischemia and infarction in the lower part of the left ventricle. The myocardial infarction model ligated by ligating the left anterior descending branch of the coronary artery in the mouse heart was divided into 4 experimental groups. The 4 experimental groups were injected with the reprogramming factor mRNA preparation of Example 1, the eGFP protein mRNA preparation of Example 2, the preparation of Comparative Example 2, and the preparation of Comparative Example 9 at multiple points (2-3 points) in the myocardial ischemia and infarction area, and then the sternum and pectoral muscle of the mouse were sutured to ensure that the wound was firmly closed. The fifth experimental group (sham operation group) used healthy mice that had undergone sham operation. After the reprogramming factor mRNA preparation of Example 1 was injected into the myocardium at multiple points (2-3 points) in the myocardial area, the sternum and pectoral muscles of the mice were sutured to ensure that the wound was firmly closed.

[0156] 2. Forced clicking on the treadmill to evaluate the improvement of cardiac function after treatment with the reprogramming factor mRNA preparation of Example 1, the eGFP protein mRNA preparation of Example 2, the preparation of Comparative Example 2, the preparation of Comparative Example 9, and the sham operation group. The treadmill was not started, and the mice to be tested rested and adapted for 30 minutes. The speed was started at 5 m / min with a 10% slope and increased by 5 m / min every 5 minutes until it reached 25 m / min. The total time was 36.8 minutes and the total distance was 507.4 meters. The proportion of mice tolerating the treadmill, the persistence time, and the running distance were evaluated.

[0157] 3. Fibrosis staining after myocardial infarction in the five experimental groups (reprogramming factor mRNA preparation of Example 1, eGFP protein mRNA preparation of Example 2, preparation of Comparative Example 2, preparation of Comparative Example 9, sham operation group) was performed. The mouse hearts were removed and fixed with 4% PFA. The fixed hearts were dehydrated and immersed so that they could be cut before slicing. Dehydration was performed using gradually increasing alcohol concentrations (such as 70%, 80%, 95%, 100%), and then the hearts were immersed in an appropriate tissue embedding agent (such as wax or freezing medium). The embedded hearts were cut into thin slices, usually 4-8 microns thick. Cutting was performed using a microtome or a slicing blade. The slices were transferred to glass slides, and the heart slices were immersed in Picrosirius Red dye, and then the excess dye was washed off with an acidic solution. Finally, the slices were observed under a microscope. The collagen fibers appeared red or orange, and Image J was used to quantify the extent and proportion of fibrosis.

[0158] Experimental results and analysis:

[0159] As shown in Figures 3 to 9, the mRNA preparation of the present application rapidly expresses reprogramming factors to improve damaged heart function. As shown in Figures 3 to 7, the therapeutic effect of the reprogramming factor mRNA preparation of Example 1 was evaluated by forced electric shock treadmill evaluation of the myocardial infarction model of the left anterior descending coronary artery ligated mouse heart. Compared with the reporter gene treatment group of Example 2, the reprogramming factor mRNA preparation of Example 1 significantly improved cardiac function after myocardial infarction treatment; please refer to Figure 4, the preparation of Example 1 showed a significantly higher proportion of mice with myocardial infarction that could tolerate forced electric shock treadmill than the preparation of Example 2; please refer to Figure 5, the preparation of Example 1 could tolerate forced electric shock treadmill for a significantly longer time than the preparation of Example 2; please refer to Figure 6, the preparation of Example 1 showed a significantly longer forced electric shock treadmill distance than the preparation of Example 2; please refer to Figure 7, the preparation of Example 1 significantly reduced the scar area after myocardial infarction than the preparation of Example 2.

[0160] Referring to Figure 8 , comparing the reporter gene mRNA preparation of Example 2 and the VEGFA mRNA preparation of Comparative Example 9, the reprogramming factor mRNA preparation-treated group of Example 1 showed a significant upregulation in the expression of Ki67, an indicator of cardiomyocyte proliferation, while the reporter gene mRNA preparation of Example 2 and the VEGFA mRNA preparation of Comparative Example 9 had no effect on cardiomyocyte proliferation.

[0161] As shown in Figure 9, the myocardial injury indicator cTNT expression did not change significantly in the reporter gene mRNA preparation group of Example 2 and the VEGFA mRNA preparation group of Comparative Example 9, while the reprogramming factor mRNA preparation group of Example 1 was treated. This indicates that the reprogramming factor mRNA preparation of Example 1 exerts its protective effect by promoting cardiomyocyte proliferation rather than protecting cardiomyocytes. In contrast, the myocardial injury indicator cTNT was significantly improved in the comparative reporter gene mRNA preparation group of Example 2 and the VEGFA protein mRNA preparation group of Comparative Example 9, demonstrating the myocardial protective effect of the VEGFA mRNA preparation of Comparative Example 9.

[0162] Example 5:

[0163] The eGFP protein mRNA preparation of Example 2 was injected into the skin of mice. The corresponding eGFP protein mRNA of Example 2 was injected into the skin, i.e., between the epidermis and dermis. At the corresponding time points, the fluorescence intensity was measured using a small animal imaging device.

[0164] The eGFP protein mRNA preparation of Comparative Example 12 was injected into the skin of mice. After the corresponding eGFP protein mRNA preparation of Comparative Example 12 was injected into the skin, that is, between the epidermis and dermis, the fluorescence intensity was measured by a small animal imaging instrument at the corresponding time points.

[0165] Example 6:

[0166] The eGFP protein mRNA preparation of Example 2 was injected into the mouse muscle. The corresponding eGFP protein mRNA of Example 2 was injected into the mouse hind leg muscle. At the corresponding time points, the fluorescence intensity was measured using a small animal imaging instrument.

[0167] The eGFP protein mRNA preparation of Comparative Example 12 was injected into the mouse muscle. The corresponding eGFP protein mRNA of Comparative Example 12 was injected into the mouse hind leg muscle. At the corresponding time points, the fluorescence intensity was measured by a small animal imaging instrument.

[0168] Experimental results and analysis:

[0169] Please refer to Figures 10 to 12 , which show that the mRNA preparation of Example 2 of the present application is efficiently expressed in the skin, muscles, and bone joint cavities.

[0170] As shown in Figure 10 , the eGFP protein mRNA formulation of Example 2 was rapidly and efficiently expressed 3 hours after intradermal injection in mice (A, corresponding to Example 5) and 3 hours after intramuscular injection (B, corresponding to Example 6). Referring to Figure 11 , the eGFP protein mRNA formulation of Example 2 was rapidly and efficiently expressed in the bone and joint cavity, with significant expression at 24 hours and continued high expression at 96 hours. The eGFP protein mRNA formulation of Comparative Example 12 showed weak expression at 24 hours and no expression at 96 hours, demonstrating that the mRNA formulation of Example 2 was highly advantageous compared to the formulation of Comparative Example 12.

[0171] Example 7:

[0172] The eGFP protein mRNA preparation of Example 2 was injected into the bone joint cavity, and the reprogramming factor mRNA preparation of Example 1 was injected into the bone joint cavity after iodoacetic acid cartilage injury to restore cartilage regeneration. SD rats were selected and anesthetized with isoflurane. Before the operation, the skin of the rat's surgical area was disinfected. The skin of the rat was carefully incised with a scalpel, and the soft tissue was separated to expose the knee joint or cartilage. A fine needle tip or microtweezers was used to create multiple stimulation points on the cartilage surface. Iodoacetic acid solution was applied to the cartilage injury area. The mice were divided into two experimental groups. The two experimental groups were respectively injected with the reprogramming factor mRNA preparation of Example 1 and the eGFP protein mRNA preparation of Example 2 into the damaged cartilage tissue. The soft tissue and skin were sutured to ensure that the wound was firmly closed. After 5 weeks, the cartilage tissue was taken and stained with cinnamon orange G and HE. The third experimental group (sham operation group) used healthy mice with sham operation. The reprogramming factor mRNA preparation of Example 1 was injected into the cartilage tissue. After 5 weeks, the cartilage tissue was taken and stained with cinnamon orange G and HE.

[0173] Specific steps:

[0174] 1. Fix cartilage tissue: Remove the cartilage tissue to be studied and fix the tissue using 4% PFA fixative.

[0175] 2. Embedding: Dehydrate the fixed cartilage tissue for embedding in paraffin. Place the tissue in increasing concentrations of dehydrated alcohol (70%, 95%, and 100%), maintaining each concentration for 5 minutes to ensure adequate dehydration. Then, immerse the tissue in a clearing agent, benzene glue, to make it transparent.

[0176] 3. Embedding cartilage tissue: Place the transparent cartilage tissue in melted paraffin and allow it to solidify into a paraffin block.

[0177] 4. Sectioning: Use a tissue slicer to cut the cartilage paraffin block into very thin sections with a thickness of 5 μm.

[0178] 5. Degreasing: Immerse the cartilage slices in a degreasing agent to remove the paraffin in the slices for subsequent staining.

[0179] 6. Cinnamon Orange G staining: Place the defatted cartilage sections in Cinnamon Orange G staining solution for 5 minutes.

[0180] 7. Cleaning: Clean the stained sections appropriately to remove excess dye.

[0181] 8. Dehydration and sealing: Gradually immerse the cartilage slices in decreasing concentrations of dehydrated alcohol, then transfer them to a transparent agent, and finally cover them on a glass slide and cover the slices with a sealing medium to seal them.

[0182] 9. Microscopic observation: Under the microscope, cartilage appears red, and cell nuclei and collagen fibers appear blue. Video and HE staining: First, tissue sections fixed and embedded in 4% PFA need to be obtained, and then dehydrated and embedded to obtain sections. Dewaxing and dehydration: Immerse the sections of the wax block in the wax remover Xylene to remove the wax.

[0183] Washing: Wash the dewaxed sections under running tap water to thoroughly remove any remaining dewaxing agent. Staining: Immerse the sections in a series of stains, starting with a hematoxylin solution, which stains the cell nucleus and surrounding material. The sections are then transferred to an eosin solution, which stains the cytoplasm and extracellular matrix.

[0184] Dehydration: Dehydrate the stained sections by sequentially immersing them in different concentrations of alcohol (e.g., 70% alcohol, 95% alcohol, and absolute alcohol). Detergent Treatment: Immerse the sections in an appropriate detergent, such as Xylene or other detergents, to remove the alcohol and render them transparent. Mounting: Place the sections in an appropriate medium, such as a transparent mounting medium (e.g., Mounting Medium), between a glass slide and a cover slip, then cover with a coverslip.

[0185] After completing the above steps, the sections are ready. Using a microscope, the HE-stained sections can be used to observe the staining of cell nuclei (blue or purple), cytoplasm and extracellular matrix (pink). Image J software is used to quantify cartilage thickness, and Graphpad Prism software is used for statistics and data output.

[0186] Experimental results and analysis:

[0187] As shown in Figures 13 and 14, the reprogramming factor mRNA preparation of Example 1 of the present application is efficiently expressed in the rat bone joint cavity to restore damaged cartilage regeneration. After the iodoacetic acid destruction cartilage model (osteoarthritis model) was successfully established, the GFP mRNA preparation treatment group of Comparative Example 2 (B in Figure 13) was treated with silica orange G staining to indicate that the reprogramming factor mRNA preparation of Example 1 significantly increased the thickness of cartilage, and repaired after iodoacetic acid destruction of cartilage (C in Figure 13), and substantially returned to normal levels (A in Figure 13). Refer to Figure 14, which is a comparison of the results of the corresponding group of rat cartilage thickness for Image J quantitative output, and the results are consistent with observation under a microscope.

[0188] Example 8: Evaluation of the therapeutic efficacy of different ratios of reprogramming factor combinations on myocardial therapy

[0189] This example is divided into six experimental groups:

[0190] Experimental group 1 (empty preparation treatment group): using the preparation of Comparative Example 2;

[0191] Experimental group 2 (eGFP mRNA preparation treatment group): using the eGFP protein mRNA preparation of Example 2;

[0192] Experimental group 3 (VEGF mRNA preparation treatment group): using the preparation of Comparative Example 9;

[0193] Experimental group 4 (OSKGL (3:1:1:1:1) mRNA preparation-treated group): using the preparation of Example 1;

[0194] Experimental group 5 (OSKGL (1:1:1:1:1) mRNA preparation-treated group): the preparation of Comparative Example 10 was used;

[0195] Experimental group 6 (OSKGL (1:1:1:1:1) mRNA preparation-treated group): the preparation of Comparative Example 11 was used;

[0196] A myocardial infarction model was established in mice by ligating the left anterior descending coronary artery as in Example 4. Different experimental groups were treated, and Horvath epigenetic clock assay (https: / / horvath.genetics.ucla.edu / html / dnamage / ) was performed at corresponding time points. Post-myocardial infarction fibrosis staining was performed as in Example 4, and the size of the post-myocardial infarction scar was assessed using Image J.

[0197] Experimental Results and Analysis: As shown in Figure 15 , experimental group 4 significantly reduced Horvath apparent age and the area of ​​post-infarction scars compared to experimental groups 2, 1, 5, and 6. The above description is merely an embodiment of the present application. It should be noted that those skilled in the art can make improvements without departing from the inventive concept of the present application, and such improvements are within the scope of protection of the present application.

Claims

1. A tissue cell proliferation reprogramming factor preparation, characterized in that: The invention comprises 7 to 8 molar parts of mRNA nucleic acid molecules encoding mutant replicase of alphavirus, 2 to 4 molar parts of mRNA nucleic acid molecules encoding Oct4 transcription factor, 1 molar part of mRNA nucleic acid molecules encoding Sox2 transcription factor, 1 molar part of mRNA nucleic acid molecules encoding Klf4 transcription factor, 1 molar part of mRNA nucleic acid molecules encoding Glis1 transcription factor and 1 molar part of mRNA nucleic acid molecules encoding Lin28 transcription factor.

2. The tissue cell proliferation promoting reprogramming factor preparation according to claim 1, characterized in that: It includes 7 to 8 molar parts of mRNA nucleic acid molecules encoding mutant alphavirus replicase, 3 molar parts of mRNA nucleic acid molecules encoding Oct4 transcription factor, 1 molar part of mRNA nucleic acid molecules encoding Sox2 transcription factor, 1 molar part of mRNA nucleic acid molecules encoding Klf4 transcription factor, 1 molar part of mRNA nucleic acid molecules encoding Glis1 transcription factor and 1 molar part of mRNA nucleic acid molecules encoding Lin28 transcription factor.

3. The tissue cell proliferation promoting reprogramming factor preparation according to claim 1, characterized in that: The tissue cell proliferation promoting reprogramming factor preparation also includes a first solvent, the concentration of citrate in the first solvent is 0-15 mmol / L, and the concentration of sodium chloride in the first solvent is 120-140 mmol / L.

4. The tissue cell proliferation promoting reprogramming factor preparation according to claim 3, characterized in that: The concentration of citrate in the first solvent is 10 mmol / L, the concentration of sodium chloride is 130 mmol / L, and the concentration of sucrose is 0-0.1 g / mL, and the pH value of the first solvent is 7.5; Alternatively, the concentration of citrate in the first solvent is 10 mmol / L, the concentration of sodium chloride is 130 mmol / L, and the concentration of sodium dihydrogen phosphate is 0-2 mmol / L, and the pH value of the first solvent is 7.

5.

5. The tissue cell proliferation promoting reprogramming factor preparation according to claim 1, characterized in that: Each of the mRNA nucleic acid molecules comprises a 5' cap structure, a 5' UTR sequence, a coding sequence of a corresponding transcription factor mRNA or a coding sequence of an alphavirus mutant replicase mRNA, a 3' UTR sequence and a polyadenylation sequence.

6. The tissue cell proliferation promoting reprogramming factor preparation according to claim 1, characterized in that: Some or all of the nucleotides in the mRNA nucleic acid molecule encoding the mutant alphavirus replicase, the mRNA nucleic acid molecule encoding the Oct4 transcription factor, the mRNA nucleic acid molecule encoding the Sox2 transcription factor, the mRNA nucleic acid molecule encoding the Klf4 transcription factor, the mRNA nucleic acid molecule encoding the Glis1 transcription factor, and the mRNA nucleic acid molecule encoding the Lin28 transcription factor have been chemically modified to improve the stability of the mRNA nucleic acid molecules in vivo.

7. The tissue cell proliferation promoting reprogramming factor preparation according to claim 6, characterized in that: The chemical modification includes replacing 100% of the cytosine in the mRNA nucleic acid molecules encoding the mutant replicase of the alphavirus genus, the mRNA nucleic acid molecules encoding the Oct4 transcription factor, the mRNA nucleic acid molecules encoding the Sox2 transcription factor, the mRNA nucleic acid molecules encoding the Klf4 transcription factor, the mRNA nucleic acid molecules encoding the Glis1 transcription factor, and the mRNA nucleic acid molecules encoding the Lin28 transcription factor with 5-methylcytosine, and replacing 100% of the uracil in the mRNA nucleic acid molecules encoding the mutant replicase of the alphavirus genus, the mRNA nucleic acid molecules encoding the Oct4 transcription factor, the mRNA nucleic acid molecules encoding the Sox2 transcription factor, the mRNA nucleic acid molecules encoding the Klf4 transcription factor, the mRNA nucleic acid molecules encoding the Glis1 transcription factor, and the mRNA nucleic acid molecules encoding the Lin28 transcription factor with N1-methylpseudouridine.

8. The tissue cell proliferation promoting reprogramming factor preparation according to claim 1, characterized in that: The mutant replicase produces a mutation at position 259 of the nsP2 region and a mutation at position 650 of the nsP2 region, wherein the mutation at position 259 of the nsP2 region is from serine S to proline P, and the mutation at position 650 of the nsP2 region is from arginine R to aspartic acid D.

9. Use of the tissue cell proliferation reprogramming factor preparation according to any one of claims 1 to 8 in promoting cardiomyocyte proliferation or in preparing cardiac gene therapy drugs.

10. Use of the tissue cell proliferation reprogramming factor preparation according to any one of claims 1 to 8 in promoting chondrocyte proliferation or in preparing a bone and joint repair drug.

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