CN116870197A - Protissue cell proliferation reprogramming factor preparation and application - Google Patents
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- CN116870197A CN116870197A CN202311026909.XA CN202311026909A CN116870197A CN 116870197 A CN116870197 A CN 116870197A CN 202311026909 A CN202311026909 A CN 202311026909A CN 116870197 A CN116870197 A CN 116870197A
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Abstract
The embodiment of the application provides a pro-tissue cell proliferation reprogramming factor preparation and application, wherein the pro-tissue cell proliferation reprogramming factor preparation comprises mRNA for coding a alphavirus mutant replicase, mRNA for coding an Oct4 transcription factor, mRNA for coding a Sox2 transcription factor, mRNA for coding a Klf4 transcription factor, mRNA for coding a Glis1 transcription factor and mRNA for coding a Lin28 transcription factor; through the mode, the molar ratio of mRNA expressing replicase and mRNA nucleic acid molecules of different transcription factors is adjusted, after the reprogramming factor preparation is introduced into the myocardium, the synthesis of each transcription factor is realized through the alphavirus mutant replicase, so that myocardial cells can be promoted to enter cell division and increase myocardial cell number, the cardiac function of a damaged heart is improved, and particularly, the cardiac function of acute cardiac injury is improved; meanwhile, uncontrolled growth of myocardial cells is avoided, and hypertrophic cardiomyopathy and heart failure are avoided.
Description
Technical Field
The application relates to the technical field of biological medicines, in particular to a reprogramming factor preparation for promoting tissue cell proliferation and application thereof.
Background
The treatment of heart failure is a clinically significant unmet need, with significant increases in disability and mortality in heart failure patients over the last 20 years, and up to date there is a lack of therapies to alter the course of heart failure, so how to find new effective treatments is one of the most significant subjects in medicine in this century.
Loss of cardiomyocytes and/or loss of function are the pathological basis of heart failure, and coronary bypass and in situ heart transplantation have provided new approaches to heart failure treatment in recent years, but post-treatment cardiac fibrosis and donor serious shortages have limited their clinical use.
The new cardiac cell transplantation method can supplement new cardiac cells with contractile function to the damaged heart so as to improve the function of the damaged heart, but the cardiac cells are terminal differentiated cells, adult cardiac cells completely lose the division capacity, so that the clinical application of the cardiac cell transplantation method is greatly limited because the cardiac cells with the contractile capacity cannot be provided.
Furthermore, the field of cardiac gene therapy is advancing, but it has not been widely used clinically. Currently, the most widely used gene therapy is achieved by viral vectors, in particular adeno-associated viral (AAV) vectors. Over the past few decades, adenoviruses, related adenoviruses (AAV), lentiviruses, and DNA plasmids have been used to introduce genes of interest into cardiomyocytes. AAV and adenovirus both have high myocardial transfection levels, whereas lentivirus and DNA plasmid have low myocardial transfection efficiency. However, adenovirus can cause a strong immune response, and is difficult to be clinically applied, so AAV is the only suitable option for the current cardiac gene therapy. However, the pharmacokinetics of AAV in the heart (expression starting from day 4 and remaining for at least 11 months) may lead to uncontrolled growth of the myocardium, hypertrophic cardiomyopathy and heart failure. Moreover, over 60% of healthy human individuals have neutralizing antibodies against AAV capsids, which can effectively neutralize gene expression delivered by this method, which drawbacks limit the use of AAV in cardiac gene therapy. More serious are: delivery of the protein of interest via AAV may be associated with a severe immune response (allergy) to AAV.
Currently, there is a need to propose a new cardiac gene therapy drug without risk of genome integration.
Disclosure of Invention
In view of the above problems, embodiments of the present application provide a circuit, a chip, and an electronic device, so as to solve the above technical problems.
In a first aspect, embodiments of the present application provide a pro-tissue cell proliferation reprogramming factor formulation 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.
Alternatively, 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 are included.
Optionally, the pro-tissue cell proliferation reprogramming factor preparation further comprises a first solvent, wherein 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.
Optionally, the concentration of citrate in the first solvent is 10mmol/L, the concentration of sodium chloride is 130mmol/L and the concentration of sucrose is 0-0.1 g/mL, and the pH value of the first solvent is 7.5;
or the concentration of citrate in the first solvent is 10mmol/L, the concentration of sodium chloride is 130mmol/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 pro-tissue cell proliferation reprogramming factor formulation of claim 1, wherein each of the mRNA nucleic acid molecules comprises a 5' cap structure, a 5' utr sequence, a coding sequence for a corresponding transcription factor mRNA or a coding sequence for an alphavirus mutant replicase mRNA, a 3' utr sequence, and a polyadenylation sequence.
Optionally, a part or all of the nucleotides in the mRNA nucleic acid molecule encoding the mutant replicase of the genus EmRNAs, 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 molecule in vivo.
Alternatively, the chemical modification comprises replacing 100% of the cytosines in the mRNA nucleic acid molecule encoding the alphavirus mutant 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-methylcytidine, and replacing 100% of the uracils in the mRNA nucleic acid molecule encoding the alphavirus mutant 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.
Alternatively, the mutant replicase produces a mutation at position 259 of the nsP2 region which is a mutation at position 259 of the nsP2 region to proline P and a mutation at position 650 of the nsP2 region which is a mutation at arginine R to aspartic acid D.
In a second aspect, embodiments of the present application provide an application of the above-mentioned pro-tissue cell proliferation reprogramming factor preparation in promoting myocardial cell proliferation or in preparing a cardiac gene therapy drug.
In a third aspect, the present application provides an application of the above-mentioned pro-tissue cell proliferation reprogramming factor preparation in promoting chondrocyte proliferation or preparing a bone joint repair drug. .
The embodiment of the application provides a pro-tissue cell proliferation reprogramming factor preparation and application, comprising 7-8 mole parts of mRNA nucleic acid molecule for encoding alpha virus mutant replicase, 2-4 mole parts of mRNA nucleic acid molecule for encoding Oct4 transcription factor, 1 mole part of mRNA nucleic acid molecule for encoding Sox2 transcription factor, 1 mole part of mRNA nucleic acid molecule for encoding Klf4 transcription factor, 1 mole part of mRNA nucleic acid molecule for encoding Glis1 transcription factor and 1 mole part of mRNA nucleic acid molecule for encoding Lin28 transcription factor; through the mode, the molar ratio of mRNA expressing replicase and mRNA nucleic acid molecules of different transcription factors is regulated, a specific tissue cell proliferation reprogramming factor preparation is formed, after the reprogramming factor preparation is introduced into cardiac muscle, the alpha virus mutant replicase is synthesized, the synthesis of each transcription factor is realized through the alpha virus mutant replicase, myocardial cells can be promoted to enter cell division, the number of myocardial cells is increased, and then the cardiac function of a damaged heart, in particular the cardiac function of acute cardiac injury is improved; meanwhile, uncontrolled growth of myocardial cells is avoided, and hypertrophic cardiomyopathy and heart failure are avoided.
These and other aspects of the application will be more readily apparent from the following description of the embodiments.
Drawings
FIG. 1 shows a graph of fluorescein intensity versus injection time for example 3 of the present application.
FIG. 2 shows a bar graph of fluorescein intensity versus the intensity for example 3 of the present application.
Fig. 3 shows a comparative plot of the experimental site of example 4 of the present application.
Fig. 4 is a comparative diagram showing the proportion of mice with myocardial infarction of the forced electric shock resistant treadmill of example 4 of the present application.
Fig. 5 shows a comparison of time for a force shock tolerant treadmill of example 4 of the present application.
Figure 6 shows a comparison of forced shock treadmill distance versus the present application of example 4.
FIG. 7 shows a graph of scar area contrast after myocardial infarction according to example 4 of the present application.
FIG. 8 is a graph showing the proliferation of cardiomyocytes according to example 4 of the present application.
Fig. 9 shows a comparative graph of the myocardial performance protecting effect of example 4 of the present application.
Fig. 10 shows a comparison of the expression of the different experimental groups in example 5 and example 6 of the present application.
Fig. 11 shows a comparison of the bone joint cavity expression changes in different experimental groups of examples 5 and 6 according to the present application.
FIG. 12 shows a comparison of IFN-a changes from different experimental groups of example 5 and example 6 of the present application.
Fig. 13 shows a microscopic comparison of cartilage thickness in example 7 of the present application.
FIG. 14 is a graph showing the comparison of the results of the measurement of the thickness of cartilage in example 7 of the present application.
FIG. 15 is a graph showing the results of evaluating the effect of different ratio reprogramming factor combinations on myocardial therapy in example 8 of the present application.
Detailed Description
Embodiments of the present application are described in detail below, examples of which are illustrated in the accompanying drawings, wherein like or similar reference numerals refer to like or similar elements or elements having like or similar functions throughout. The embodiments described below by referring to the drawings are exemplary only for explaining the present application and are not to be construed as limiting the present application.
In order to enable those skilled in the art to better understand the solution of the present application, the following description will make clear and complete descriptions of the technical solution of the present application in the embodiments of the present application with reference to the accompanying drawings. It will be apparent that the described embodiments are only some, but not all, embodiments of the application. All other embodiments, which can be made by those skilled in the art based on the embodiments of the application without making any inventive effort, are intended to be within the scope of the application.
In the embodiments of the present application, it should be noted that, in this document, relational terms such as first and second, and the like are used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions.
Moreover, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus. Without further limitation, an element defined by the phrase "comprising one … …" does not exclude the presence of other like elements in a process, method, article, or apparatus that comprises the element.
In describing embodiments of the present application, words such as "exemplary" or "such as" are used to mean illustrated, described, or described. Any embodiment or design described as "exemplary" or "such as" in an embodiment of the application is not necessarily to be construed as preferred or advantageous over another embodiment or design. The use of words such as "example" or "such as" is intended to present relative concepts in a clear manner.
In addition, the term "plurality" in the embodiments of the present application means two or more, and in view of this, the term "plurality" may be understood as "at least two" in the embodiments of the present application. "at least one" may be understood as one or more, for example as one, two or more. For example, including at least one means including one, two or more, and not limiting what is included, e.g., including at least one of A, B and C, then A, B, C, A and B, A and C, B and C, or A and B and C, may be included.
It should be noted that, in the embodiment of the present application, "and/or" describe the association relationship of the association object, which means that three relationships may exist, for example, a and/or B may be represented: a exists alone, A and B exist together, and B exists alone. The character "/", unless otherwise specified, generally indicates that the associated object is an "or" relationship.
The experimental methods in the following examples are conventional methods unless otherwise specified.
Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.
An embodiment of the application provides a pro-tissue cell proliferation reprogramming factor preparation comprising 7-8 mole parts of mRNA nucleic acid molecule encoding an alpha virus mutant replicase, 2-4 mole parts of mRNA nucleic acid molecule encoding an Oct4 transcription factor, 1 mole part of mRNA nucleic acid molecule encoding a Sox2 transcription factor, 1 mole part of mRNA nucleic acid molecule encoding a Klf4 transcription factor, 1 mole part of mRNA nucleic acid molecule encoding a Glis1 transcription factor, and 1 mole part of mRNA nucleic acid molecule encoding a Lin28 transcription factor.
In this example, by adjusting the molar ratio of mRNA nucleic acid molecules expressing different transcription factors, a specific pro-tissue cell proliferation reprogramming factor preparation is formed, after the myocardium is introduced with the reprogramming factor preparation, an alphavirus mutant replicase is synthesized, and the synthesis of each transcription factor is realized by the alphavirus mutant replicase, so that cardiomyocytes can be promoted to enter cell division and the number of cardiomyocytes can be increased, and further, the cardiac function of a damaged heart, in particular, the cardiac function of acute cardiac injury can be improved; meanwhile, uncontrolled growth of myocardial cells is avoided, and hypertrophic cardiomyopathy and heart failure are avoided.
As one embodiment, the pro-tissue cell proliferation reprogramming factor formulation comprises 7 to 8 molar parts of mRNA nucleic acid molecule encoding an alphavirus mutant replicase, 3 molar parts of mRNA nucleic acid molecule encoding an Oct4 transcription factor, 1 molar part of mRNA nucleic acid molecule encoding a Sox2 transcription factor, 1 molar part of mRNA nucleic acid molecule encoding a Klf4 transcription factor, 1 molar part of mRNA nucleic acid molecule encoding a Glis1 transcription factor, and 1 molar part of mRNA nucleic acid molecule encoding a Lin28 transcription factor.
As one embodiment, the preparation of the reprogramming factor for promoting tissue cell proliferation further comprises a first solvent, wherein 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. The above-described mRNA nucleic acid molecule encoding an alpha-viral mutant replicase, mRNA nucleic acid molecule encoding an Oct4 transcription factor, mRNA nucleic acid molecule encoding a Sox2 transcription factor, mRNA nucleic acid molecule encoding a Klf4 transcription factor, mRNA nucleic acid molecule encoding a Glis1 transcription factor, and mRNA nucleic acid molecule encoding a Lin28 transcription factor are dissolved in a first solvent, which may be formed from a citrate and physiological saline configuration.
In this embodiment, each mRNA nucleic acid molecule can be made less susceptible to degradation by the first solvent.
In some embodiments, the first solvent has a citrate concentration of 10mmol/L, sodium chloride concentration of 130mmol/L, and sucrose concentration of 0-0.1 g/mL, and the first solvent has a pH of 7.5;
in some embodiments, the first solvent has a citrate concentration of 10mmol/L, a sodium chloride concentration of 130mmol/L, and a sodium dihydrogen phosphate concentration of 0-2 mmol/L, and a pH of 7.5.
In some embodiments, the first solvent has a pH of 7.5 and has a citrate concentration of 10mmol/L, a sodium chloride concentration of 130mmol/L, a sodium dihydrogen phosphate concentration of 0-2 mmol/L, and a sucrose concentration of 0-0.1 g/mL.
As one embodiment, each mRNA nucleic acid molecule includes a 5' cap structure, a 5' utr sequence, a coding sequence for a corresponding transcription factor mRNA or a coding sequence for an alphavirus mutant replicase mRNA, a 3' utr sequence, and a polyadenylation sequence.
Specifically, mRNA nucleic acid molecules encoding the alphavirus mutant replicase include a 5' cap structure, a 5' utr sequence, an alphavirus mutant replicase mRNA coding sequence, a 3' utr sequence, and a polyadenylation sequence; mRNA nucleic acid molecules encoding the Oct4 transcription factor include 5' cap structure, 5' UTR sequence, mRNA coding sequence for the Oct4 transcription factor, 3' UTR sequence and polyadenylation sequence; mRNA nucleic acid molecules encoding Sox2 transcription factors include 5' cap structure, 5' UTR sequence, mRNA coding sequence for Sox2 transcription factor, 3' UTR sequence and polyadenylation sequence; mRNA nucleic acid molecules encoding the Klf4 transcription factor include 5' cap structure, 5' UTR sequence, mRNA coding sequence for the Klf4 transcription factor, 3' UTR sequence, and polyadenylation sequence; mRNA nucleic acid molecules encoding the Glis1 transcription factor include 5' cap structure, 5' UTR sequence, mRNA coding sequence for the Glis1 transcription factor, 3' UTR sequence and polyadenylation sequence; mRNA nucleic acid molecules encoding the Lin28 transcription factor include 5' cap structure, 5' UTR sequence, mRNA coding sequence for the Lin28 transcription factor, 3' UTR sequence, and polyadenylation sequence.
As one embodiment, a part or all of the nucleotides in the mRNA nucleic acid molecule encoding the alphavirus mutant 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 molecule in vivo.
In some embodiments, the chemical modification comprises replacing 100% of the cytosines in the mRNA nucleic acid molecule encoding the alphavirus mutant 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-methylcytidine, and replacing 100% of the uracils in the mRNA nucleic acid molecule encoding the alphavirus mutant 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.
In one embodiment, the mutant replicase produces a mutation at position 259 of the nsP2 region wherein serine S is mutated to proline P and a mutation at position 650 of the nsP2 region wherein arginine R is mutated to aspartic acid D.
In this embodiment, after the above-described reprogramming factor preparation is introduced into the myocardium, the alphavirus mutant replicase is synthesized, and the synthesis of each transcription factor is realized by the alphavirus mutant replicase, so that the limited self-replication of each transcription factor can be realized, the uncontrolled growth of cardiomyocytes can be avoided, the occurrence of hypertrophic cardiomyopathy and heart failure can be avoided, and the safety of the reprogramming factor preparation can be improved.
In other embodiments, the reprogramming factor preparations described above may not include an mRNA nucleic acid molecule encoding an alphavirus mutant replicase, and each transcription factor may be synthesized using replicase in cardiomyocytes.
The embodiment of the application also provides the application of the tissue cell proliferation promoting reprogramming factor preparation in promoting myocardial cell proliferation or preparing cardiac gene therapy medicines.
The preparation of the reprogramming factors is introduced by using a myocardial intramuscular injection method, and each reprogramming factor is expressed through cells of the heart of the organism, so that the reprogramming of the epigenetic part is started, and the epigenetic expression of the myocardial cells is readjusted, so that the epigenetic state of the myocardial cells is reversed and is more similar to a young state, and the repair and the function improvement of the damaged heart are rapidly promoted.
The embodiment of the application also provides the application of the tissue cell proliferation promoting reprogramming factor preparation in promoting chondrocyte proliferation or preparing a bone joint repair drug.
Among them, the inventors of the present application have unexpectedly found that the reprogramming factor preparation can be efficiently expressed in the osteoarticular cavity, skin and muscle, and can restore the regeneration of damaged cartilage.
Example 1: preparation of reprogramming factor preparation for promoting tissue cell proliferation
A) Preparation of mRNA encoding an alpha-viral mutant replicase
Step one, utilize GeneArtTM GibsonHiFi reaction (Thermo Fisher, U.S.A.A.46624) synthesizes a mutant replicase DNA coding sequence (DNA nucleic acid molecule encoding an alpha-viral mutant replicase and does not contain a polyadenylation sequence), and after successful synthesis, the mutant replicase DNA coding sequence is cloned into pcDNA3.3 vector plasmid for commercial production.
1.1 mutant replicase DNA coding sequence: the 5 'untranslated region DNA sequence (SEQ ID NO. 9), the mutant replicase coding sequence and the 3' untranslated region DNA sequence (SEQ ID NO. 10), wherein the mutant replicase coding sequence is divided into four DNA fragments, namely a nsP1 region fragment (SEQ ID NO. 3), a nsP2 region fragment (SEQ ID NO. 4), a nsP3 region fragment (SEQ ID NO. 5) and a nsP4 region fragment (SEQ ID NO. 6), and the four DNA fragments are all modified fragments with high GC content. Four DNA fragments were directly ordered as gblock from IDT company, USA.
The method specifically comprises the following steps: the Gibson reaction was assembled according to Table 1 and reacted in a PCR apparatus at 50℃for 60 minutes to obtain a PCR product.
TABLE 1 Gibson reaction System
1.2 transformation of PCR products into One Shottm TOP10 chemocompetent E.coli cells, comprising the following steps:
diluting the Gibson reaction system (PCR product) with water without nuclease according to a ratio of 1:5, uniformly mixing 12 mu L of water without nuclease with 3 mu LGibson reaction system, and carrying out ice reaction;
adding 1 mu L of the diluent into One Shottm TOP10 chemically competent E.coli cells and mixing, and incubating the transformation mixture on ice for 20-30 minutes;
incubating the cells at 42 ℃ for 30 seconds without shaking;
the reaction tube was immediately transferred to ice and incubated on ice for 2 minutes;
mu.L of room temperature S.O.C. broth (US Life Technology) was added;
shaking at 300rpm at 37℃for 1 hour;
100. Mu.L of the plated bacterial culture plate (100. Mu.g/mL of ampicillin or 50. Mu.g/mL of kanamycin.);
bacterial clones were selected overnight at 37 ℃, shaken at 37 ℃ and the double mutant replicase sequence plasmids containing the correct sequences were selected for the first-generation sequencing.
Step two, adding poly- (a) tail of mRNA by PCR to obtain DNA synthesis template of replicase mRNA
Wherein the poly- (a) tail comprises 120 adenylates.
PCR premix (total volume 200. Mu.L, 25. Mu.L for each of the eight reactions) was prepared according to Table 2;
TABLE 2 composition of PCR premix
PCR reactions were performed according to the reaction conditions shown in Table 3;
TABLE 3 PCR reaction conditions
| Number of cycles | Denaturation (denaturation) | Annealing | Expansion of |
| 1 | 95℃,2–3min | ||
| 2-31 | 98℃,20s | 60℃,15s | 72℃,60s |
| 32 | 72℃,3min |
Checking the quality of the PCR products by gel electrophoresis;
the PCR product was recovered by gel cutting (QIAquick PCR purification kit, qiagen, cat.no.28106) and the final concentration of the tail template was adjusted to 100 ng/. Mu.L as a DNA synthesis template for in vitro transcription synthesis of mRNA encoding replicase.
Step three, in vitro transcription synthesis of mRNA encoding replicase
1. mRNA cap structures and nucleotide mixtures were assembled according to table 4:
hat Structure 3'-O-Me-m7G (5') ppp (5 ') G RNA cap analogue (New England Biolabs, cat.no. S1411S), -methyytidine-5' -triphosphite (Me-CTP; trilink, cat.no. N1014), N1-methyl-pseudo-UTP (Trilink, cat.no. N1019), the other components were all from MEGAscript T7 kit (Ambion, cat.no. AM1334).
TABLE 4 mRNA cap structure and nucleotide mixture
2. The in vitro transcription system encoding replicase mRNA was assembled according to table 5:
TABLE 5 in vitro transcription System for mRNA encoding replicase
| Component (A) | Dosage (ml) | Final concentration |
| DNase/RNase-free water | 1.2 | |
| Custom NTP(from last step) | 14.8 | |
| Tailed PCR product,100ug/μL | 16 | 40ng/μL |
| T7 buffer,10X((from MEGAscript T7 kit) | 4.0 | 1X |
| T7 enzyme mix,10×(from MEGAscript T7 kit) | 4.0 | 1X |
3. The reaction is placed in a PCR instrument and incubated for 3 to 6 hours at 37 ℃.
4. To each sample 2 μl of Turbo DNase (from MEGAscript T7 kit, ambion, cat.no. am 1334) was added.
5. Mix gently and incubate at 37℃for 15min.
6. The reaction treated with DNase and RNAa seIII was purified using the MEGAclear kit (Ambion, cat.no. AM1908); the modified mRNA was eluted with a total of 100. Mu.L of elution buffer (50. Mu.L of elution buffer was eluted twice).
7. Purified modified mRNA was treated with phosphatase (Antarctic phosphatase (New England Biolabs, cat.no. M0289S).
8. To each sample (-100. Mu.L) 11. Mu.L of 10 Xphosphatase buffer was added followed by 2. Mu.L of phosphatase; the samples were gently mixed and incubated at 37℃for 0.5-1 h.
9. After elution, the concentration of modified replicase-encoding mRNA was measured in a NanoDrop spectrophotometer. The expected total yield should be-50 ug (30-70 ug range; 100 ul elution volume of one 40 ul IVT reaction is 300-700 ng ul). The concentration was adjusted to 100 ng/. Mu.L by adding elution buffer or TE buffer (pH 7.0), or FPLC purification.
B) Preparation of mRNA encoding each transcription factor
The synthesis procedure of mRNA encoding transcription factor is similar to that of mRNA encoding replicase, and includes the following steps:
step one, utilize GeneArtTM Gibson HiFi reaction (Thermo Fisher, U.S.A.A.46624) synthesizes a specifically modified target factor DNA coding sequence (the nucleic acid molecule encoding the transcription factor mRNA does not contain a polyadenylation sequence);
wherein the specifically modified target factor DNA coding sequence: a 5 'untranslated region DNA sequence (SEQ ID NO. 9), a replicase 5' end specific DNA sequence (SEQ ID NO. 7), a target factor DNA coding sequence (see Table 6), a replicase 3 'end specific DNA sequence (SEQ ID NO. 8), a 3' untranslated region DNA sequence (SEQ ID NO. 10).
Step two, adding poly- (a) tail of mRNA on the target factor DNA coding sequence of the specific modification through PCR to obtain a DNA synthesis template for coding the transcription factor mRNA;
and thirdly, in vitro transcription synthesis of mRNA encoding the transcription factor.
The 5 kinds of mRNA encoding transcription factors shown in Table 6 were synthesized according to the above-described method.
TABLE 6 DNA synthesis templates for mRNA encoding different transcription factors
C) mRNA encoding the mutant replicase of the genus A, 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 were dissolved in a molar ratio of 7:3:1:1:1:1:1 in a first solvent (10 mmol/L citrate, 130mmol/L sodium chloride, 0.15g/mL sucrose nuclease-free distilled water, pH 7.5).
Example 2: preparation of different mRNAs encoding fluorescent proteins
The synthesis procedure of mRNA encoding fluorescent protein is similar to that of mRNA encoding replicase, and comprises the following steps:
step one, utilize GeneArtTM Gibson HiFi reaction (Thermo Fisher, U.S.A.A.46624) synthesizes a specifically modified fluorescent protein DNA coding sequence (nucleic acid molecules encoding fluorescent protein mRNA do not contain polyadenylation sequences);
wherein, the specific modified fluorescent protein DNA coding sequence: a 5 'untranslated region DNA sequence (SEQ ID NO. 9), a replicase 5' end specific DNA sequence (SEQ ID NO. 7), a fluorescent protein DNA coding sequence of interest (see Table 7), a replicase 3 'end specific DNA sequence (SEQ ID NO. 8), a 3' untranslated region DNA sequence (SEQ ID NO. 10).
Step two, adding the poly- (a) tail of mRNA on a target fluorescent protein DNA coding sequence subjected to specific modification by PCR to obtain a DNA synthesis template for coding fluorescent protein mRNA;
and thirdly, in vitro transcription synthesis of mRNA for encoding fluorescent protein.
The 2 kinds of mRNA encoding transcription factors shown in Table 7 were synthesized according to the above-described method.
TABLE 7 DNA synthesis templates for different mRNAs encoding fluorescent proteins
Example 2-eGFP protein mRNA preparation: mRNA encoding the mutant replicase of the genus alpha virus and mRNA encoding the eGFP protein were dissolved in a first solvent (10 mmol/L citrate, 130mmol/L sodium chloride, 0.15g/mL sucrose nuclease-free distilled water, pH 7.5).
Example 2-firefly luciferin protein mRNA preparation: mRNA encoding the mutant alpha-viral replicase and mRNA encoding firefly luciferin protein were dissolved in a first solvent (10 mmol/L citrate, 130mmol/L sodium chloride, 0.15g/mL sucrose nuclease free distilled water, pH 7.5).
Comparative example 1: common firefly luciferin protein mRNA preparation
mRNA encoding eGFP protein was dissolved in a first solvent (10 mmol/L citrate, 130mmol/L sodium chloride, 0.15g/mL sucrose nuclease free distilled water, pH 7.5).
Comparative example 2: empty mRNA preparation
First solvent (10 mmol/L citrate, 130mmol/L sodium chloride, 0.15g/mL sucrose nuclease free distilled water, pH 7.5).
Comparative example 3: mRNA encoding mutant replicase of the genus A and mRNA encoding firefly luciferin protein were dissolved in physiological saline.
Comparative example 4: mRNA encoding the mutant replication enzyme of the genus Emamectin and mRNA encoding the firefly luciferin protein were dissolved in a second solvent (10 mmol/L citrate, 130mmol/L sodium chloride, pH 7.5).
Comparative example 5:
mRNA encoding the mutant replicase of the genus Emamectin and mRNA encoding the firefly luciferin protein in a solvent in calcium phosphate solution.
Comparative example 6:
mRNA encoding the mutant replicase of the genus A and mRNA encoding the firefly luciferin protein were solubilized in RNAimax transfection reagent (Life Technologies).
Comparative example 7:
mRNA encoding the mutant replicase of the genus EmV and mRNA encoding the firefly luciferin protein were dissolved in Invivo JetPEI transfection reagent (Polyplus).
Comparative example 8:
mRNA encoding the mutant replicase of the genus Emamectin and mRNA encoding the firefly luciferin protein were solubilized in MC3 nanoliposomes (Cayman Chem).
Comparative example 9:
the synthesis procedure of mRNA encoding vegfa protein is similar to that of mRNA encoding replicase, and comprises the following steps:
step one, utilize GeneArtTM GibsonHiFi reaction (Thermo Fisher, U.S.A.A 46624) synthesizes a specifically modified vegfa protein DNA coding sequence (nucleic acid molecules encoding the vegfa protein mRNA do not contain a polyadenylation site)A nucleotide sequence);
wherein the specific modified vegfa protein DNA coding sequence: a 5 'untranslated region DNA sequence (SEQ ID NO. 9), a replicase 5' end specific DNA sequence (SEQ ID NO. 7), a vegfa protein DNA coding sequence, a replicase 3 'end specific DNA sequence (SEQ ID NO. 8), a 3' untranslated region DNA sequence (SEQ ID NO. 10).
Step two, adding the poly- (a) tail of mRNA to the specially modified vegfa protein DNA coding sequence by PCR to obtain a DNA synthesis template for coding the vegfa protein mRNA;
and thirdly, in vitro transcription synthesis of mRNA encoding the vegfa protein.
The preparation comprises mRNA encoding the mutant replicase of the genus alpha virus and mRNA encoding the vegfa protein, dissolved in a first solvent (10 mmol/L citrate, 130mmol/L sodium chloride, 0.15g/mL sucrose nuclease free distilled water, pH 7.5).
Comparative example 10:
mRNA encoding the mutant replicase of the genus A, 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 were dissolved in a molar ratio of 7:1:1:1:1:1:1:1 in a first solvent (10 mmol/L citrate, 130mmol/L sodium chloride, 0.15g/mL sucrose nuclease-free distilled water, pH 7.5).
Comparative example 11:
mRNA encoding the mutant replicase of the genus A, 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 were dissolved in a molar ratio of 7:0.5:1:1:1:1 in a first solvent (10 mmol/L citrate, 130mmol/L sodium chloride, 0.15g/mL sucrose nuclease free distilled water, pH 7.5).
Comparative example 12:
mRNA encoding the mutant replicase of the genus alphavirus and mRNA encoding the eGFP protein were solubilized in MC3 nanoliposomes (Cayman Chem).
Example 3: expression imaging experiments
The expression of the firefly fluorescent protein (Luci) mRNA preparation was quantitatively detected by the IVIS imager by the intramuscular injection verification of the mRNA preparation of the mouse heart-specific reporter green fluorescent protein (eGFP) or firefly luciferin protein, specifically by exposing the left chest region of the mouse, cutting the chest, revealing the heart, dividing into 9 experimental groups, the 9 experimental groups injecting the reporter-modified mRNA preparation of example 2 described above in the free myocardium of the left ventricle of the mouse, respectively, the normal 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 invitvo jetpi delivery preparation of comparative example 7, the MC3 nanoliposomes of comparative example 8, and the limited self-replication m RNA intramyocardial injections of comparative examples 5 to 8 were performed according to the manufacturer's instructions, respectively.
Experimental results and analysis:
Referring to fig. 1 and 2, the firefly luciferin protein mRNA preparation of example 2 of the present application is highly expressed intramyocardially, and as shown in fig. 1, the time of peak of single limited self-replication firefly luciferin protein mRNA preparation of example 2 is 480 hours after injection, the duration of firefly luciferin protein expression is 1300 hours after injection, and both the peak and duration of expression of example 2 are significantly improved compared to the common firefly luciferin mRNA preparation of comparative example 1.
As shown in fig. 2, the formulation of example 2 showed the highest expression of the reporter limited self-replication mRNA compared to the other formulation components (comparative examples 3 to 8), and the non-liposomal delivery reporter limited self-replication mRNA showed significantly higher expression than the liposomal delivery reporter mRNA in cardiac intramuscular injection applications.
Referring to FIG. 12, the formulation of example 2 showed no significant increase in IFN-a in the serum of mice 24 hours after injection compared to the formulation of comparative example 8 after cardiac intramyocardial injection, indicating that the mRNA formulation of example 2 showed no immune response to MC3 nanoparticles.
Example 4: myocardial treatment effect evaluation experiment
Mouse cardiac coronary artery left anterior descending ligation myocardial infarction model establishment and preparation treatment and efficacy evaluation of example 1. The method comprises the following specific steps:
1, establishing a mouse heart coronary artery left anterior descending ligation myocardial infarction model: selecting a suitable C57 mouse, isoflurane anesthesia, sterilizing the chest of the mouse prior to the beginning of the surgery, carefully exposing the heart by making a midline incision in the chest of the mouse, using microsurgery techniques and a magnifying glass, finding the left anterior descending branch (left anterior descending artery) of the mouse heart, ligating it with absorbable threads, where the ligation is located near the coronary artery, which would result in left ventricular lower myocardial ischemia and infarction, dividing the mouse heart coronary artery left anterior descending branch ligature myocardial infarction model into 4 experimental groups, dividing the reprogramming factor mRNA formulation of example 1, the eGFP protein mRNA formulation of example 2, the formulation of comparative example 2, and the formulation of comparative example 9 after multi-point (2-3 points) intramyocardial injection in the myocardial ischemia and infarction area, suturing the sternum and pectoral muscle of the mouse, ensuring firm closure of the wound. The 5 th experimental group (sham-operated group) used sham-operated healthy mice, and after injecting the reprogramming factor mRNA preparation of example 1 into the myocardium at a plurality of points (2-3 points) in the myocardial region, the sternum and pectoral muscles of the mice were sutured to ensure firm closure of the wound.
2 forced clicking on the treadmill to evaluate the reprogramming factor mRNA formulation of example 1, the eGFP protein mRNA formulation of example 2, the formulation of comparative example 9, cardiac function improvement after sham treatment, the treadmill not activated, resting and acclimatizing the mice to be tested 30min,10% gradient, increasing 5m/min every 5min at a 5m/min activation rate until 25m/min, total time 36.8min, total distance 507.4m, evaluation of the mouse tolerance treadmill ratio, holding time, distance traveled, respectively.
3, five experimental groups described above (reprogramming factor mRNA formulation of example 1, eGFP protein mRNA formulation of example 2, formulation of comparative example 9, sham surgery group) were stained for fibrosis after heart infarction, mouse hearts were removed and 4% pfa was immobilized, and the immobilized hearts were dehydrated and impregnated so as to be able to be cut before slicing. The heart is dehydrated using progressively higher alcohol concentrations (e.g., 70%, 80%, 95%, 100%) and then immersed in a suitable tissue embedding medium (e.g., wax or freezing medium) to slice the embedded heart into thin slices, typically 4-8 microns thick. The cut is made using a microtome or a microtome blade. Sections were transferred onto glass slides, heart sections were immersed in picrosides Red dye, excess dye was then washed off with an acidic solution, and finally sections were observed using a microscope, collagen fibers were Red or orange, and Image J quantified for fibrosis range and ratio.
Experimental results and analysis:
as shown in fig. 3 to 9, the mRNA preparation of the present application rapidly expresses reprogramming factors to improve impaired cardiac function, and as shown in fig. 3 to 7, the treatment effect evaluation of the reprogramming factor mRNA preparation of example 1 was performed by evaluating a left anterior descending coronary artery ligation cardiac myocardial infarction model of a mouse heart by a forced electric shock treadmill, and the cardiac function after myocardial infarction was significantly improved by comparing the reporter gene treatment group of example 2 with the reprogramming factor mRNA preparation of example 1; referring to fig. 4, the formulation of example 1 exhibited a significantly higher proportion of mice with myocardial infarction in the forced shock-tolerant treadmill than the formulation of example 2; referring to fig. 5, the formulation of example 1 was significantly longer than the formulation of example 2 to withstand a forced shock treadmill; referring to fig. 6, the formulation of example 1 is significantly longer than the formulation of example 2, which forces a shocking treadmill; referring to fig. 7, the formulation of example 1 showed significantly smaller scar area after myocardial infarction than the formulation of example 2.
Referring to fig. 8, the treatment of the reporter mRNA preparation of comparative example 2 and the VEGFA mRNA preparation of comparative example 9, the reprogramming factor mRNA preparation treatment of example 1, the expression of the cardiomyocyte proliferation index Ki67 was significantly up-regulated, whereas the reporter mRNA preparation of example 2 and the VEGFA mRNA preparation of comparative example 9 had no effect on the proliferation of cardiomyocytes.
Referring to fig. 9, the treatment of the reporter mRNA preparation of example 2 and the VEGFA mRNA preparation of comparative example 9, the reprogramming factor mRNA preparation treatment of example 1 showed no significant change in myocardial damage index cTNT expression, demonstrating that the reprogramming factor mRNA preparation of example 1 exerts a protective effect by promoting cardiomyocyte proliferation rather than by protecting cardiomyocytes, whereas the VEGFA mRNA preparation of example 2 and the VEGFA protein mRNA preparation of comparative example 9 showed significant improvement in myocardial damage index cTNT, showing the effect of the VEGFA mRNA preparation of comparative example 9 on protecting myocardium.
Example 5:
the gfp protein mRNA preparation of example 2 was injected into the skin of mice, and the fluorescence intensity was measured by a small animal imager at a corresponding time point after the corresponding gfp protein mRNA of example 2 was injected into the skin, i.e., between the epidermis and the dermis.
The gfp protein mRNA preparation of comparative example 12 was injected into the skin of mice, and after the corresponding gfp protein mRNA preparation of comparative example 12 was injected into the skin, i.e., between the epidermis and the dermis, the fluorescence intensity was measured by a small animal imager at the corresponding time point.
Example 6:
mice were injected intramuscularly with the eGFP protein mRNA preparation of example 2, and the fluorescence intensity was measured by a small animal imager at the corresponding time point after the injection of the corresponding eGFP protein mRNA of example 2 into the back leg muscle of the mice.
Mice were injected intramuscularly with the preparation of the eGFP protein mRNA of comparative example 12, and after the corresponding eGFP protein mRNA of comparative example 12 was injected into the back leg muscle of the mice, the fluorescence intensity was measured by a small animal imager at the corresponding time point.
Experimental results and analysis:
referring to FIGS. 10 to 12, the mRNA preparation of example 2 of the present application was expressed efficiently in the cavities of skin, muscle and bone joints.
As shown in FIG. 10, the eGFP protein mRNA preparation of example 2 was rapidly and efficiently expressed 3 hours after the intradermal injection (A, corresponding to example 5) and 3 hours after the intramuscular injection (B, corresponding to example 6) in mice. Referring to FIG. 11, the eGFP protein mRNA preparation of example 2 was expressed rapidly and efficiently in the bone joint cavity, with significant expression for 24 hours and still high expression for 96 hours; the eGFP protein mRNA formulation of comparative example 12 had weaker expression at 24 hours and no expression at 96 hours; the mRNA formulation of example 2 is illustrated to be highly advantageous compared to the formulation of comparative example 12.
Example 7:
bone joint cavity injection of the eGFP protein mRNA formulation of example 2 was performed and cartilage regeneration was resumed by injection of the reprogramming factor mRNA formulation of example 1 after iodoacetic acid cartilage injury. SD rats were selected for isoflurane anesthesia, skin sterilization was performed on the surgical area of the rats before the operation was started, the skin of the rats was carefully incised using a scalpel, soft tissue was isolated to expose knee joints or cartilage, a plurality of stimulation points were created on the cartilage surface using a fine needle tip or micro forceps, an iodoacetic acid solution was applied to the cartilage damaged area, the mice were divided into two experimental groups, soft tissue and skin were sutured after injection of the reprogramming factor mRNA preparation of example 1 and the eGFP protein mRNA preparation of example 2 on the damaged cartilage tissue, ensuring firm closure of the wound, and cartilage tissue was taken after 5 weeks with concurrent cinnamon lime G staining HE staining. The third experimental group used healthy mice (sham-operated group) with sham surgery, the reprogramming factor mRNA preparation of example 1 was injected into cartilage tissue, and cartilage tissue was taken 5 weeks later and stained with cinnamon lime G staining HE.
The method comprises the following specific steps:
1, fixing cartilage tissue: cartilage tissue to be studied was removed and the tissue was fixed using 4% pfa fixative.
2, embedding: the fixed cartilage tissue is dehydrated so as to be embedded in paraffin. Cartilage tissue was gradually placed in increasing concentrations of dehydrated alcohol (70%, 95%, 100% ethanol), each concentration maintained for a period of 5 minutes to ensure adequate dehydration. Then, the tissue was immersed in a transparent agent benzene gum to make it transparent.
3, embedding cartilage tissue: the transparentized cartilage tissue is placed in melted paraffin and allowed to solidify into paraffin blocks.
4, slicing: the cartilage paraffin block was cut into very thin sections, 5 microns thick, using a tissue microtome.
5, degreasing: the cartilage sections were immersed in a degreasing agent to remove paraffin from the sections for subsequent staining.
6, cinnamon lime G staining: the defatted cartilage sections were placed in cinnamon lime G staining solution for 5 minutes.
7, cleaning: the stained sections were suitably washed to remove excess dye.
8, dehydrating and sealing: the cartilage slices were gradually immersed in decreasing concentrations of dehydrated alcohol, then transferred to a transparentizing agent, finally covered on glass slides and the slices were covered with a capper to seal them.
9, microscopic observation: cartilage appears red under microscope, nuclei and collagen fibers appear blue, and HE staining is performed with imaging: firstly, obtaining a tissue slice fixed and embedded by 4% PFA, and finally obtaining the slice through dehydration and embedding; dewaxing and dehydrating: the slices of the wax block were immersed in a dewaxing agent Xylene to remove waxes.
Washing: the dewaxed slice is washed under running tap water to thoroughly remove the dewaxed agent residue. Dyeing: the sections are sequentially immersed in a stain, first a heme stain (hemadyloxy) which stains the cell nucleus and the material surrounding the cell nucleus. The sections were then transferred to an acid wine red (Eosin) solution, which stained the cytosol and extracellular matrix.
Dehydrating: the stained sections were sequentially immersed in different concentrations of alcohol (e.g., 70% alcohol, 95% alcohol, and absolute alcohol) to dehydrate the sections. And (3) cleaning agent treatment: the slices are immersed in a suitable cleaning agent, such as Xylene or other cleaning agent, to remove alcohol and make the slices transparent. Sealing piece: the sections are placed in a suitable Medium, such as a clear coverslip (e.g., a moving Medium) between a glass slide and a cover slip, and then covered with a glass slide cover slip.
After the above steps are completed, the slice is ready. Using a microscope, the staining of the nuclei (blue or purple) and the staining of the cytosol and extracellular matrix (pink) can be observed by HE-stained sections, image J software for thickness quantification of cartilage, and Graphpad Prism software for statistics and data output.
Experimental results and analysis:
as shown in fig. 13 and 14, the reprogramming factor mRNA preparation of example 1 of the present application was highly expressed in the joint space of rat bone to restore damaged cartilage regeneration. After the successful establishment of the iodoacetic acid-disrupted cartilage model (osteoarthritis pattern), the GFP mRNA preparation treatment group of comparative example 2 (B in fig. 13), orange G silicate staining suggested that the reprogramming factor mRNA preparation of example 1 significantly increased the thickness of cartilage, achieved the repair after the iodoacetic acid-disrupted cartilage (C in fig. 13), and restored to substantially normal levels (a in fig. 13). Referring to fig. 14, the Image J quantitatively outputs the corresponding set of rat cartilage thickness results, which are consistent with the microscopic observation.
Example 8: evaluation of myocardial treatment efficacy by different ratio reprogramming factor combinations
The present example is divided into six experimental groups:
experimental group 1 (empty formulation treatment group): the formulation of comparative example 2 was used;
experimental group 2 (eGFP mRNA formulation treatment group): using the eGFP protein mRNA formulation of example 2;
experimental group 3 (VEGF mRNA formulation treatment group): the formulation of comparative example 9 was used;
experimental group 4 (OSKGL (3:1:1:1:1) mRNA formulation treatment group): the formulation of example 1 was used;
experimental group 5 (OSKGL (1:1:1:1:1) mRNA formulation treatment group): the formulation of comparative example 10 was used;
Experimental group 6 (OSKGL (1:1:1:1:1) mRNA formulation treatment group): the formulation of comparative example 11 was used;
a model of mouse cardiac coronary artery left anterior descending ligation myocardial infarction was established as in example 4, treatment was performed using different experimental groups, horvath apparent clock detection (https:// Horvath. Genetics. Ucla. Edu/html/dnamage /) was performed at corresponding time points, and post-cardiac infarction fibrosis staining was performed as in example 4, and Image J assessed the size of the scar after cardiac infarction.
Experimental results and analysis: as shown in fig. 15, the apparent age of Horvath is obviously reduced and the area of scar after heart infarction is obviously reduced in the experimental group 4 compared with the experimental group 2, the experimental group 1, the experimental group 5 and the experimental group 6. While the application has been described with respect to the above embodiments, it should be noted that modifications can be made by those skilled in the art without departing from the inventive concept, and these are all within the scope of the application.
Claims (10)
1. A pro-tissue cell proliferation reprogramming factor formulation comprising 7-8 molar parts of mRNA nucleic acid molecule encoding an alphavirus mutant replicase, 2-4 molar parts of mRNA nucleic acid molecule encoding an Oct4 transcription factor, 1 molar part of mRNA nucleic acid molecule encoding a Sox2 transcription factor, 1 molar part of mRNA nucleic acid molecule encoding a Klf4 transcription factor, 1 molar part of mRNA nucleic acid molecule encoding a Glis1 transcription factor, and 1 molar part of mRNA nucleic acid molecule encoding a Lin28 transcription factor.
2. The pro-tissue cell proliferation reprogramming factor formulation of claim 1, comprising 7-8 molar parts of mRNA nucleic acid molecule encoding a alphavirus mutant replicase, 3 molar parts of mRNA nucleic acid molecule encoding an Oct4 transcription factor, 1 molar part of mRNA nucleic acid molecule encoding a Sox2 transcription factor, 1 molar part of mRNA nucleic acid molecule encoding a Klf4 transcription factor, 1 molar part of mRNA nucleic acid molecule encoding a Glis1 transcription factor, and 1 molar part of mRNA nucleic acid molecule encoding a Lin28 transcription factor.
3. The pro-tissue cell proliferation reprogramming factor formulation of claim 1, further comprising a first solvent, wherein 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 pro-tissue cell proliferation reprogramming factor formulation according to claim 3, wherein the concentration of citrate in the first solvent is 10mmol/L, the concentration of sodium chloride is 130mmol/L and the concentration of sucrose is 0-0.1 g/mL, the pH of the first solvent is 7.5;
or the concentration of citrate in the first solvent is 10mmol/L, the concentration of sodium chloride is 130mmol/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 pro-tissue cell proliferation reprogramming factor formulation of claim 1, wherein each of the mRNA nucleic acid molecules comprises a 5' cap structure, a 5' utr sequence, a coding sequence for a corresponding transcription factor mRNA or a coding sequence for an alphavirus mutant replicase mRNA, a 3' utr sequence, and a polyadenylation sequence.
6. The pro-tissue cell proliferation reprogramming factor formulation of claim 1, wherein some or all of the nucleotides in the mRNA nucleic acid molecule encoding the alphavirus mutant 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 increase the stability of the mRNA nucleic acid molecule in vivo.
7. The pro-tissue cell proliferation reprogramming factor formulation of claim 6, wherein the chemical modification comprises replacing 100% of cytosine in the mRNA nucleic acid molecule encoding the alphavirus mutant replicase, the mRNA nucleic acid molecule encoding the Oct4 transcription factor, the mRNA nucleic acid molecule encoding Sox2 transcription factor, the mRNA nucleic acid molecule encoding Klf4 transcription factor, the mRNA nucleic acid molecule encoding Glis1 transcription factor and the mRNA nucleic acid molecule encoding Lin28 transcription factor with 5-methylcytidine, and replacing 100% of uracil in the mRNA nucleic acid molecule encoding the alphavirus mutant replicase, the mRNA nucleic acid molecule encoding Oct4 transcription factor, the mRNA nucleic acid molecule encoding Sox2 transcription factor, the mRNA nucleic acid molecule encoding Klf4 transcription factor, the mRNA nucleic acid molecule encoding Glis1 transcription factor and the mRNA nucleic acid molecule encoding Lin28 transcription factor with N1-methylpseudouridine.
8. The pro-tissue cell proliferation reprogramming factor formulation of claim 1, wherein the mutant replicase produces a mutation at position 259 of the nsP2 region and a mutation at position 650 of the nsP2 region, the mutation at position 259 of the nsP2 region is serine S to proline P, and the mutation at position 650 of the nsP2 region is arginine R to aspartic acid D.
9. Use of a formulation of a pro-tissue cell proliferation reprogramming factor according to any of claims 1 to 8 for promoting cardiomyocyte proliferation or for the preparation of a cardiac gene therapy medicament.
10. Use of a formulation of a pro-tissue cell proliferation reprogramming factor according to any of claims 1 to 8 for promoting chondrocyte proliferation or for the preparation of a bone joint repair medicament.
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