WO2024207617A1

WO2024207617A1 - Reprogramming factor anti-senility mrna composition, preparation method therefor and use thereof

Info

Publication number: WO2024207617A1
Application number: PCT/CN2023/099480
Authority: WO
Inventors: 王刚; 于寅; 盛强龙; 杨帆
Original assignee: 臻赫医药(杭州)有限公司
Priority date: 2023-04-03
Filing date: 2023-06-09
Publication date: 2024-10-10
Also published as: CN116474120A

Abstract

A reprogramming factor anti-senility mRNA composition, a preparation method therefor and the use thereof. Using chemically modified mRNAs as non-virus gene delivery vectors avoids inherent potential safety hazards due to using virus vectors, avoids inherent potential safety hazards of tumorigenesis of DNA vectors since mRNAs do not enter cell nucleuses and do not integrate genomes, and additionally helps to reduce costs. The chemically modified mRNAs are transiently expressed, which helps to control the in-vivo half-life period, thereby improving the anti-senility effect. Adjusting the molar ratio of a first mRNA to a second mRNA achieves the proportional control of different reprogramming factors, thereby improving the anti-senility effect.

Description

Reprogramming factor anti-aging mRNA composition, preparation method and application

Technical Field

The present application relates to the field of biomedicine technology, and in particular to a reprogramming factor anti-aging mRNA composition, a preparation method and an application thereof.

Background Art

The loss of epigenetic information is one of the main driving forces of the occurrence, development and outcome of aging. Studies have shown that reprogramming factors in cells can restore lost epigenetic information through epigenetic reprogramming, thereby partially reversing the aging of organs such as skin, heart, kidney, brain, osteoporosis, ovaries and testicles, improving the ability to repair organ damage and extending the lifespan of experimental animals. This is a major conceptual breakthrough in biology.

However, there are very strict requirements for the degree and duration of epigenetic reprogramming for effective anti-aging, so there is a gap between the "epigenetic reprogramming anti-aging concept" and "conversion into clinical practice means", which is still basically blank. The reasons are as follows: First, epigenetic reprogramming anti-aging needs to find a balance between effectiveness and safety: the degree of epigenetic reprogramming for anti-aging needs to be between 40%-60% (average 57%), too little is not enough to achieve the anti-aging effect, too much will cause changes in cell properties and thus cancer. Secondly, the time of epigenetic reprogramming must be intermittent and continuous, and it cannot last too long at a time, otherwise it will exceed 60% of epigenetic reprogramming and cause changes in cell properties and cancer. Thirdly, traditional anti-aging treatment has the sole goal of improving the overall function of the body and prolonging life, and the effect is difficult or impossible to quantify. The in situ anti-aging of specific organs can be quantitatively evaluated by the effect of specific organ function and post-injury repair, and then achieve the goal of improving the overall function of the body and prolonging life. Therefore, the in situ anti-aging of specific organs is highly beneficial compared with traditional anti-aging, but the in situ anti-aging research of specific organs is basically blank.

In the background technology, the only in situ anti-aging case at the specific organ level is: in a mouse model, using AAV (adeno-associated virus) viral vectors encoding Oct4, Sox2, and Klf4 to partially reverse optic nerve aging in situ in the eyeball. However, the above method still cannot bridge the gap between "epigenetic remodeling" and "epigenetic remodeling". There is a gap between the concept of "epijping anti-aging" and "conversion into clinical practice". This method has insurmountable obstacles in the safety and effectiveness of epigenetic reprogramming anti-aging. The above method has the following inherent obstacles: First, AAV viral vectors, no matter how artificially modified, have viral properties, such as a certain incidence of lethal allergies, and the risk of tumors caused by genome integration, which is its inherent, traditional safety hazard. Secondly, the expression time of AAV viral vector AAV encoding Oct4, Sox2, Klf4 reprogramming factors is mostly more than one year. Such a long-term continuous expression of Oct4, Sox2, Klf4 reprogramming factors will lead to Otherwise, it will exceed 60% of epigenetic reprogramming, causing changes in cell properties and cancer, and new safety risks of AAV viral vectors. Thirdly, the host exposed to AAV viral vectors for the first time will produce antibodies against AAV within a few weeks, making it impossible to reuse AAV as a vector for gene therapy. The basic premise of epigenetic anti-aging is to repeatedly and wave-like epigenetic reprogramming to achieve anti-aging effects. Therefore, the epigenetic reprogramming of Oct4, Sox2, and Klf4 encoded by AAV viral vectors has limited effectiveness in anti-aging. Finally, AAV viral vector treatment is expensive, and the cost of a course of treatment is over one million US dollars.

Summary of the invention

The purpose of this application is to provide a reprogramming factor anti-aging mRNA composition, preparation method and application to solve the above-mentioned technical problems.

In a first aspect, the present application provides a reprogramming factor anti-aging mRNA composition, comprising:

The first mRNA encoding the Oct4 transcription factor;

at least one second mRNA encoding a target reprogramming factor;

Part or all of the nucleotides in the first mRNA or the second mRNA are chemically modified to improve the stability of the first mRNA or the second mRNA in vivo, and the molar ratio of the first mRNA to the second mRNA is 2-8.

Optionally, the target reprogramming factor is a Sox2 transcription factor, a Klf4 transcription factor, a c-Myc transcription factor, a Lin28 transcription factor or a Glis1 transcription factor.

Optionally, part or all of the uracil in the first mRNA or the second mRNA is chemically modified to improve the stability of the first mRNA or the second mRNA in vivo, wherein the chemical modification comprises replacing the first mRNA with N1-methylpseudouridine, pseudouracil or methyluracil. At least 50% uracil in the mRNA or the second mRNA.

Optionally, part or all of the cytosines in the first mRNA or the second mRNA are chemically modified to improve the stability of the first mRNA or the second mRNA in an organism, and the chemical modification includes replacing at least 50% of the cytosines in the first mRNA or the second mRNA with 5-methylcytosine or 5-hydroxymethylcytosine.

Optionally, the chemical modification includes replacing 100% of uracil in the first mRNA or the second mRNA with N1-methylpseudouridine, pseudouracil or methyluracil and replacing 100% of cytosine in the first mRNA or the second mRNA with 5-methylcytosine or 5-hydroxymethylcytosine.

Optionally, the first mRNA includes the following elements in order from 5' to 3' direction: 5'UTR sequence, signal peptide sequence, Oct4 transcription factor coding sequence and 3'UTR sequence;

Each of the second mRNAs includes the following elements in sequence from the 5’→3’ direction: a 5’UTR sequence, a target reprogramming factor coding sequence and a 3’UTR sequence, and each of the second mRNAs also includes a signal peptide sequence.

Optionally, the reprogramming factor anti-aging mRNA composition further comprises:

a third mRNA encoding an RNA-dependent RNA polymerase;

Wherein, the molar amount of the third mRNA is 1 to 8 times the sum of the molar amounts of the first mRNA and the second mRNA.

Optionally, the RNA-dependent RNA polymerase is an alphavirus mutant replicase, which produces a mutation at position 259 of the nsP2 region and a mutation at position 650 of the nsP2 region.

In a second aspect, the present application provides an anti-aging preparation, comprising a reprogramming factor anti-aging mRNA composition, sodium citrate dihydrate and sodium chloride, wherein the reprogramming factor anti-aging mRNA composition is the above-mentioned reprogramming factor anti-aging mRNA composition, and the ratio of the sum of the masses of the first mRNA and the second mRNA in the reprogramming factor anti-aging mRNA composition, the mass of the sodium citrate dihydrate and the mass of the sodium chloride is 1-2:1-4:4-8.

In a third aspect, the present application provides a method for preparing the above-mentioned reprogramming factor anti-aging mRNA composition, characterized in that it comprises:

Synthesize the first mRNA;

synthesizing at least one second mRNA;

In a fourth aspect, the present application provides the use of the above-mentioned reprogramming factor anti-aging mRNA composition or the above-mentioned anti-aging preparation in the preparation of cell reediting reagents, in the preparation of post-organ damage repair agents, and in the preparation of anti-aging drugs.

The reprogramming factor anti-aging mRNA composition, preparation method and application of the present application use chemically modified mRNA as a non-viral gene delivery vector, thereby avoiding the inherent safety hazards of using viral vectors, and the mRNA does not enter the cell nucleus and will not integrate into the genome, thereby avoiding the inherent tumor safety hazards of DNA vectors and helping to reduce costs. Since the chemically modified mRNA is expressed transiently, it is beneficial to control the half-life in the body and improve the anti-aging effect. By adjusting the molar ratio of the first mRNA to the second mRNA, the ratio of different reprogramming factors is controlled, thereby improving the anti-aging effect.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1a is a schematic diagram of skin expression of the anti-aging preparation of Example 1 of the present application;

FIG1b is a comparison chart of the fluorescence intensity of the target protein of the anti-aging preparation of Example 1 of the present application and the comparative aqueous solution.

FIG1c is a comparison chart of the fluorescence intensity of the target protein of the anti-aging preparation of Example 1 of the present application and the comparative aqueous solution.

FIG2 is a comparison chart of the local cardiac injection effects of the anti-aging preparation of Example 2 of the present application and a comparative aqueous solution.

FIG. 3 is a comparison chart of the iPS clone numbers of Example 2 of the present application and Comparative Example 1.

DETAILED DESCRIPTION

The following will be combined with the drawings in the embodiments of the present application to clearly and completely describe the technical solutions in the embodiments of the invention. 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 ordinary technicians in this field without creative work are within the scope of protection of this application.

Reference herein to an "embodiment" means that a particular feature, structure, or characteristic described in connection with the embodiment The phrase "in some embodiments" and "in some embodiments" may be included in at least one embodiment of the present application. The phrase appearing in various locations in the specification does not necessarily refer to the same embodiment, nor is it an independent or alternative embodiment mutually exclusive with other embodiments. It is explicitly and implicitly understood by those skilled in the art that the embodiments described herein can be combined with other embodiments.

The experimental methods in the following examples are all conventional methods unless otherwise specified.

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

mRNA Composition Examples

An embodiment of the present application provides a reprogramming factor anti-aging mRNA composition, comprising: a first mRNA and at least one second mRNA, wherein the first mRNA encodes the Oct4 transcription factor, and the second mRNA encodes the target reprogramming factor, and the target reprogramming factor is a reprogramming factor other than the Oct4 transcription factor.

In this embodiment, a second mRNA may be included.

In this embodiment, a plurality of second mRNAs may be included, and the plurality of second mRNAs respectively encode a first target reprogramming factor, a second target reprogramming factor, ..., an Nth target reprogramming factor, where N is a natural number.

Part or all of the nucleotides in the first mRNA or the second mRNA are chemically modified to improve the stability of the first mRNA or the second mRNA in vivo.

Wherein, the molar ratio of the first mRNA to the second mRNA is 2-8.

The reprogramming factor anti-aging mRNA composition of the present embodiment uses chemically modified mRNA as a non-viral gene delivery vector, thereby avoiding the inherent safety hazards of using viral vectors. The mRNA does not enter the cell nucleus and will not integrate into the genome, thereby avoiding the inherent safety hazards of tumorigenesis of DNA vectors and helping to reduce costs. Since the chemically modified mRNA is expressed transiently, it is beneficial to control the half-life in the body and improve the anti-aging effect. By adjusting the molar ratio of the first mRNA to the second mRNA, the ratio of different reprogramming factors is controlled, thereby improving the anti-aging effect.

In this embodiment, after the first mRNA and at least one second mRNA enter the cell, the replication of the Oct4 transcription factor and at least one target reprogramming factor can be achieved by the RNA-dependent RNA polymerase in the cell.

As an embodiment, when multiple second mRNAs are included, the molar ratios of different second mRNAs are The amount can be the same.

As an embodiment, the target reprogramming factor is a Sox2 transcription factor, a Klf4 transcription factor, a c-Myc transcription factor, a Lin28 transcription factor or a Glis1 transcription factor.

In some embodiments, five second mRNAs may be included, namely, a second mRNA encoding a Sox2 transcription factor, a second mRNA encoding a Klf4 transcription factor, a second mRNA encoding a c-Myc transcription factor, a second mRNA encoding a Lin28 transcription factor, and a second mRNA encoding a Glis1 transcription factor.

Furthermore, the amino acid sequence of the Oct4 transcription factor is shown in SEQ ID NO.1, the amino acid sequence of the Sox2 transcription factor is shown in SEQ ID NO.2, the amino acid sequence of the Klf4 transcription factor is shown in SEQ ID NO.3, the amino acid sequence of the c-Myc transcription factor is shown in SEQ ID NO.4, the amino acid sequence of the Lin28 transcription factor is shown in SEQ ID NO.5, and the amino acid sequence of the Glis1 transcription factor is shown in SEQ ID NO.6.

As an embodiment, part or all of the uracil in the first mRNA or the second mRNA is chemically modified to improve the stability of the first mRNA or the second mRNA in vivo, and the chemical modification includes replacing at least 50% of the uracil in the first mRNA or the second mRNA with N1-methylpseudouridine, pseudouracil or methyluracil.

Furthermore, the chemical modification comprises replacing at least 60% of the uracil in the first mRNA or the second mRNA with N1-methylpseudouridine, pseudouracil or methyluracil.

Furthermore, the chemical modification comprises replacing at least 70% of uracil in the first mRNA or the second mRNA with N1-methylpseudouridine, pseudouracil or methyluracil.

Furthermore, the chemical modification comprises replacing at least 80% of uracil in the first mRNA or the second mRNA with N1-methylpseudouridine, pseudouracil or methyluracil.

Furthermore, the chemical modification comprises replacing at least 90% of uracil in the first mRNA or the second mRNA with N1-methylpseudouridine, pseudouracil or methyluracil.

Furthermore, the chemical modification comprises replacing at least 100% of uracil in the first mRNA or the second mRNA with N1-methylpseudouridine, pseudouracil or methyluracil.

In some embodiments, some or all of the cytosines in the first mRNA or the second mRNA are modified to improve the stability of the first mRNA or the second mRNA in vivo. Chemical modification, wherein the chemical modification comprises replacing at least 50% of the cytosines in the first mRNA or the second mRNA with 5-methylcytosine or 5-hydroxymethylcytosine.

Furthermore, the chemical modification comprises replacing at least 60% of the cytosine in the first mRNA or the second mRNA with 5-methylcytosine or 5-hydroxymethylcytosine.

Furthermore, the chemical modification comprises replacing at least 70% of the cytosine in the first mRNA or the second mRNA with 5-methylcytosine or 5-hydroxymethylcytosine.

Furthermore, the chemical modification comprises replacing at least 80% of the cytosines in the first mRNA or the second mRNA with 5-methylcytosine or 5-hydroxymethylcytosine.

Furthermore, the chemical modification comprises replacing at least 90% of the cytosines in the first mRNA or the second mRNA with 5-methylcytosine or 5-hydroxymethylcytosine.

Furthermore, the chemical modification comprises replacing at least 100% of the cytosine in the first mRNA or the second mRNA with 5-methylcytosine or 5-hydroxymethylcytosine.

In this embodiment, the characteristic scheme of combining natural modified uracil nucleotides with natural modified cytosine nucleotides and replacing them at a ratio of at least 50% respectively enhances the stability of the reprogramming factor anti-aging mRNA composition and reduces the immunogenicity of the reprogramming factor anti-aging mRNA composition.

In some embodiments, the chemical modification comprises replacing 100% of uracil in the first mRNA or the second mRNA with N1-methylpseudouridine and replacing 100% of cytosine in the first mRNA or the second mRNA with 5-methylcytosine or 5-hydroxymethylcytosine.

As an embodiment, the first mRNA includes the following elements in sequence from 5' to 3' direction: a 5'UTR sequence, a signal peptide sequence, an Oct4 transcription factor coding sequence and a 3'UTR sequence.

Each of the second mRNAs includes the following elements in order from 5' to 3': 5'UTR sequence, target reprogramming factor coding sequence and 3'UTR sequence, and each of the second mRNAs also includes a signal peptide sequence. In the second mRNA, the target reprogramming factor coding sequence used is different, and the position of the signal peptide sequence may be different, and the position of the signal peptide sequence can be determined according to the target reprogramming factor coding sequence.

In this embodiment, the 5'UTR sequence includes the RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO.9, and the 3'UTR sequence includes the RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO.10.

In this embodiment, the signal peptide sequence is a nuclear localization signal peptide sequence, and the Oct4 transcription factor and each target reprogramming factor are targeted to the cell nucleus under the action of the nuclear localization signal peptide.

As an embodiment, the reprogramming factor anti-aging mRNA composition of this embodiment also includes: a third mRNA, the third mRNA encodes RNA-dependent RNA polymerase (RdRp), wherein the molar amount of the third mRNA is 1 to 8 times the sum of the molar amounts of the first mRNA and the second mRNA.

In this embodiment, the replication of the Oct4 transcription factor and at least one target reprogramming factor can be achieved by the RNA-dependent RNA polymerase encoded by the third mRNA.

In some embodiments, the RNA-dependent RNA polymerase encoded by the third mRNA is a mutant replicase of the genus Alphavirus, and the mutant replicase produces a mutation at position 259 of the nsP2 region and a mutation at position 650 of the nsP2 region. Specifically, the mutant replicase includes a nsP1 region (537 amino acids), a nsP2 region (799 amino acids), a nsP3 region (482 amino acids), and a nsP4 region (1254 amino acids) connected in sequence, and the amino acid sequence of the mutant replicase is shown in SEQ ID NO.7, and the two mutation points of the mutant replicase are respectively generated at position 796 (serine S mutates to proline P) shown in SEQ ID NO.7 and at position 1187 (arginine R mutates to aspartic acid D) shown in SEQ ID NO.7.

In this embodiment, by generating specific mutations in the alphavirus replicase and reducing the activity of the alphavirus replicase, limited self-replication of the Oct4 transcription factor and at least one target reprogramming factor can be achieved through the RNA-dependent RNA polymerase encoded by the third mRNA, thereby avoiding cytotoxicity.

In some embodiments, the first mRNA includes the following elements in order from 5' to 3' direction: 5'UTR sequence, replicase 5' end specific sequence, signal peptide sequence, Oct4 transcription factor coding sequence, replicase 3' end specific sequence and 3'UTR sequence.

Each of the second mRNAs includes the following elements in sequence from 5’→3’ direction: 5’UTR sequence, replicase 5’-end specific sequence, target reprogramming factor coding sequence, replicase 3’-end specific sequence and 3’UTR sequence, and each of the second mRNAs also includes a signal peptide sequence.

In this embodiment, the replicase 5' end specific sequence includes the RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO.11, and the replicase 3' end specific sequence includes the RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO.8 The RNA sequence corresponding to the nucleic acid sequence shown.

Anti-aging formulation examples

An embodiment of the present application provides an anti-aging preparation, comprising a reprogramming factor anti-aging mRNA composition, sodium citrate dihydrate and sodium chloride, wherein the reprogramming factor anti-aging mRNA composition is the above-mentioned reprogramming factor anti-aging mRNA composition, and the ratio of the sum of the masses of the first mRNA and the second mRNA in the reprogramming factor anti-aging mRNA composition, the mass of the sodium citrate dihydrate and the mass of the sodium chloride is 1-2:1-4:2-10.

As an embodiment, the ratio of the sum of the masses of the first mRNA and the second mRNA, the mass of the sodium citrate dihydrate, and the mass of the sodium chloride in the reprogramming factor anti-aging mRNA composition is 1-2:1-4:4-8.

In this embodiment, the anti-aging preparation can be an aqueous solution, the mass concentration of the first mRNA and the second mRNA is 1-2 mg/mL, the mass concentration of sodium citrate dihydrate is 1-4 mg/mL, the mass concentration of sodium chloride is 4-8 mg/mL, and the pH value of the aqueous solution is 6-8.

As an embodiment, when the reprogramming factor anti-aging mRNA composition in the anti-aging preparation includes a third mRNA, the molar amount of the third mRNA is 1 to 8 times the sum of the molar amounts of the first mRNA and the second mRNA.

As an embodiment, for the application of reversing skin aging, the above-mentioned anti-aging preparation aqueous solution can be injected by local injection methods such as intradermal injection to introduce 100ug to 1000ug of the first mRNA and the second mRNA. The total amount of the first mRNA and the second mRNA introduced into the skin is 100ug to 1000ug.

As an embodiment, the above-mentioned anti-aging aqueous preparation can be locally injected into the heart for the heart, injected into the bone joint cavity for the bones and joints, injected into the skin or veins for the kidneys, and injected into the cerebrospinal fluid for the spinal cord and brain, thereby achieving the function of reversing the aging of specific organs and enhancing the repair of specific organ damage.

Preparation method example

The present application embodiment provides a method for preparing a reprogramming factor anti-aging mRNA composition, comprising:

Synthesize the first mRNA;

synthesizing at least one second mRNA;

As an embodiment, synthesizing the first mRNA may include the following steps:

Providing a first DNA transcription template for a first mRNA, wherein the first DNA transcription template includes the following elements in order from 5' to 3' direction: a 5'UTR sequence, a signal peptide sequence, an Oct4 transcription factor coding sequence, and a 3'UTR sequence;

Under the action of RNA polymerase, the first DNA transcription template is transcribed in vitro to obtain a first mRNA.

In some embodiments, the in vitro transcription uses a mixture of nucleoside triphosphates and their chemical modifications as raw materials, the chemical modifications include N1-methylpseudouridine and 5-methylcytosine, or the chemical modifications include N1-methylpseudouridine and 5-hydroxymethylcytosine.

As an embodiment, synthesizing the second mRNA may include the following steps:

Providing a second DNA transcription template for a second mRNA, the second DNA transcription template includes the following elements in order from 5' to 3' direction: a 5'UTR sequence, a target reprogramming factor encoding sequence, and a 3'UTR sequence, and each second mRNA also includes a signal peptide sequence;

Under the action of RNA polymerase, the second DNA transcription template is transcribed in vitro to obtain a second mRNA.

Application Examples

The embodiments of the present application provide the use of the above-mentioned reprogramming factor anti-aging mRNA composition or the above-mentioned anti-aging preparation in the preparation of cell reediting reagents, in the preparation of post-organ damage repair agents, and in the preparation of anti-aging drugs.

In some embodiments, an ex vivo human full-thickness skin model is used to introduce the mRNA composition encoding the reprogramming factor by local injection such as intradermal injection, thereby causing the reprogramming factor to downregulate the expression of the aging gene P16 in the skin organ and the oxygen free radical gene - manganese superoxide The superoxide dismutase 2 (SOD2) gene was downregulated, the extracellular matrix rejuvenation, namely the matrix metalloproteinase-1 (MMP-1) gene, the matrix metalloproteinase-9 (MMP-9) gene was downregulated, the expression of collagenase VI I was upregulated, and the histone 3 lysine 9 trimethylation (H3K9me3) was upregulated, indicating that the chemically modified mRNA encoding the reprogramming factor successfully induced skin epigenetic reprogramming by 40-60% in situ, achieving the effect of at least partially reversing skin aging.

In some embodiments, in the left anterior descending artery ligation myocardial infarction model in adult mice, the myocardial infarction area was reduced by 50% by direct local cardiac injection in the affected area, indicating that the chemically modified mRNA encoding the reprogramming factor successfully induced epigenetic reprogramming of cardiomyocytes by 40-60% in situ, achieving the effect of at least partially reversing heart aging, prompting adult mouse cardiomyocytes to enter cell replication, and regenerating the damaged heart in situ.

In addition, epigenetic reprogramming of organs such as bones and joints, kidneys, spinal cord, ovaries, testicles, brain, and osteoporosis was induced in situ by 40-60%, achieving the effect of at least reversing the aging of the corresponding organs.

Embodiment 1:

Chemically modified mRNA encoding reprogramming factors induces in situ epigenetic reprogramming of the entire human body ex vivo, at least partially reversing skin aging in situ.

Step 1: Preparation of reprogramming factor anti-aging mRNA composition

In this embodiment, the reprogramming factor anti-aging mRNA composition includes a first mRNA encoding an Oct4 transcription factor, a second mRNA encoding a Sox2 transcription factor, a second mRNA encoding a Klf4 transcription factor, a second mRNA encoding a c-Myc transcription factor, a second mRNA encoding a Lin28 transcription factor, and a second mRNA encoding a Glis1 transcription factor.

The molar amounts of the five second mRNAs are the same, and the ratio of the molar amount of the first mRNA to the sum of the molar amounts of the five second mRNAs is 2-6.

The first mRNA includes the following elements in sequence from 5'→3' direction: 5'UTR sequence, signal peptide sequence, Oct4 transcription factor coding sequence and 3'UTR sequence; each second mRNA includes the following elements in sequence from 5'→3' direction: 5'UTR sequence, target reprogramming factor coding sequence and 3'UTR sequence, and each second mRNA also includes a signal peptide sequence.

Step 2: Prepare the anti-aging preparation solution

The mass concentration of the first mRNA and the second mRNA in the anti-aging preparation aqueous solution is 1 to 2 mg/mL. The mass concentration of sodium citrate dihydrate is 1-4 mg/mL, the mass concentration of sodium chloride is 4-8 mg/mL, and the pH value of the aqueous solution is 6-8.

Step 3: Ex vivo human skin culture

The remaining skin from abdominal surgery of 45-65 years old was used, and the fat tissue was removed within 1 hour, and the full-thickness skin was retained. The skin was trimmed into about 2 square centimeters of full-thickness skin and cultured in 10% fetal bovine serum DMEM culture medium. The culture medium level did not exceed the dermis (the epidermis did not contact the culture medium).

Step 4: Intradermal injection of treatment group

100 ug to 1000 ug of the anti-aging preparation aqueous solution containing the reprogramming factor mRNA composition is introduced into the ex vivo skin.

The control group was intradermally injected with an equal amount of a contrast solution, wherein the contrast solution included a fourth mRNA with a mass concentration of 1-2 mg/mL, sodium citrate dihydrate with a mass concentration of 1-4 mg/mL, and sodium chloride with a mass concentration of 4-8 mg/mL. The pH value of the contrast solution was 6-8. The fourth mRNA encoded mCherry fluorescent protein, and the fourth mRNA included the following elements in sequence from 5'→3' direction: a 5'UTR sequence, a signal peptide sequence, an RNA sequence corresponding to the mCherry reporter gene, and a 3'UTR sequence.

Step 5: Observe the results

On the 6th day after injection, the whole-thickness skin tissue was obtained, tissue sections were prepared as usual, and immunofluorescence staining was completed. The target proteins were: aging gene P16 protein, oxygen free radical gene-manganese superoxide dismutase 2 (SOD2) protein, extracellular matrix youth remodeling i.e. matrix metalloproteinase-1 (MMP-1) protein, matrix metalloproteinase 9 (MMP-9) protein, collagenase VII protein, histone 3 lysine 9 trimethylation (H3K9me3) protein. After taking pictures with an immunofluorescence microscope, Imagine J software was used to analyze the fluorescence intensity of the corresponding proteins.

Please refer to Figures 1a, 1b and 1c. Chemically modified mRNA preparations encoding reprogramming factors Oct4, Sox2, Klf4, cMyc, Lin28 and Glis1 can reach the full thickness of human skin after intradermal injection, and induce epigenetic reprogramming in situ in the full thickness of human skin ex vivo, and adult cardiomyocytes enter cell replication, at least partially reversing skin aging.

In this embodiment, an in vitro human full-thickness skin model is used to introduce the mRNA composition encoding the reprogramming factor by local injection such as intradermal injection, thereby causing the reprogramming factor to down-regulate the expression of the aging gene P16 of the skin organ, the oxygen free radical gene - manganese superoxide dismutase 2 (SOD2) gene, the extracellular matrix rejuvenation remodeling, i.e., the matrix metalloproteinase-1 (MMP-1) gene, and the matrix metalloproteinase-2 (MMP-3) gene. The gene of matrix metalloproteinase-9 (MMP-9) was down-regulated, while the expression of collagenase VII and the trimethylation of histone 3 lysine 9 (H3K9me3) were up-regulated, indicating that the chemically modified mRNA encoding the reprogramming factor successfully induced skin epigenetic reprogramming by 40-60% in situ, achieving the effect of at least partially reversing skin aging.

Embodiment 2:

Chemically modified mRNA encoding reprogramming factors reverses cardiac aging at least in situ in the treatment of myocardial infarction in mice.

Step 1: Preparation of reprogramming factor anti-aging mRNA composition

Step 2: Prepare the anti-aging preparation solution

The mass concentration of the first mRNA and the second mRNA in the anti-aging preparation aqueous solution is 1-2 mg/mL, the mass concentration of sodium citrate dihydrate is 1-4 mg/mL, the mass concentration of sodium chloride is 4-8 mg/mL, and the pH value of the aqueous solution is 6-8.

Step 3: Local injection into the heart

A mouse heart left anterior descending artery ligation myocardial infarction model was routinely established, and immediately afterwards, 100ug to 1000ug of the anti-aging preparation aqueous solution containing the reprogramming factor mRNA composition was locally injected into the affected heart.

The control group was locally injected with an equal amount of contrast solution into the heart, wherein the contrast solution included a fourth mRNA with a mass concentration of 1 to 2 mg/mL, sodium citrate dihydrate with a mass concentration of 1 to 4 mg/mL, and sodium chloride with a mass concentration of 4 to 8 mg/mL. The pH value of the contrast solution was 6 to 8. The fourth mRNA encoded mCherry Fluorescent protein, the fourth mRNA includes the following elements in the 5'→3' direction: 5'UTR sequence, signal peptide sequence, RNA sequence corresponding to the mCherry reporter gene and 3'UTR sequence.

Step 4: Observe the results

Samples were taken on the 49th day after injection, and conventional Masson's trichrome stain was used. Photos were taken, and the area of myocardial infarction was analyzed using Imagine J software.

Please refer to Figure 2 , chemically modified mRNA encoding cell reprogramming factors Oct4, Sox2, Klf4, cMyc, Lin28, and Glis1 induced epigenetic reprogramming in situ in an adult mouse model, adult cardiomyocytes entered cell replication, and at least partially reversed myocardial infarction.

Example 3

Step 1: Preparation of reprogramming factor anti-aging mRNA composition

The molar amounts of the five second mRNAs are the same, and the ratio of the molar amount of the first mRNA to the sum of the molar amounts of the five second mRNAs is 2-8.

Step 2: Prepare the anti-aging preparation solution

Step 3: Transfection of mouse embryonic fibroblasts

100 ug to 1000 ug of the anti-aging preparation aqueous solution containing the reprogramming factor mRNA composition is introduced into mouse embryonic fibroblasts.

Comparative Example 1

Step 1: Preparation of reprogramming factor anti-aging mRNA composition

The molar amounts of the five second mRNAs are the same, and the ratio of the molar amount of the first mRNA to the sum of the molar amounts of the five second mRNAs is 1:1.

Step 2: Prepare the anti-aging preparation solution

Step 3: Transfection of mouse embryonic fibroblasts

The results of Example 3 and Comparative Example 1 are shown in Figure 3. In Example 3, the molar ratio of the first mRNA to the second mRNA is 2-8, and in Comparative Example 1, the molar ratio of the first mRNA to the second mRNA is 1:1. Controlling the molar ratio of the two to 2-8 can significantly increase the number of iPS clones, which is beneficial to enhancing the reprogramming effect.

The above is only an implementation method of the present application. It should be pointed out that a person skilled in the art can make improvements without departing from the inventive concept of the present application, but these improvements are within the scope of protection of the present application.

Claims

A reprogramming factor anti-aging mRNA composition, characterized by comprising:

The first mRNA encoding the Oct4 transcription factor;

at least one second mRNA encoding a target reprogramming factor;

Part or all of the nucleotides in the first mRNA or the second mRNA are chemically modified to improve the stability of the first mRNA or the second mRNA in vivo, and the molar ratio of the first mRNA to the second mRNA is 2-8.
The reprogramming factor anti-aging mRNA composition according to claim 1, characterized in that the target reprogramming factor is a Sox2 transcription factor, a Klf4 transcription factor, a c-Myc transcription factor, a Lin28 transcription factor or a Glis1 transcription factor.
The reprogramming factor anti-aging mRNA composition according to claim 1 is characterized in that part or all of the uracil in the first mRNA or the second mRNA is chemically modified to improve the stability of the first mRNA or the second mRNA in vivo, and the chemical modification includes replacing at least 50% of the uracil in the first mRNA or the second mRNA with N1-methylpseudouridine, pseudouracil or methyluracil.
The reprogramming factor anti-aging mRNA composition according to claim 3 is characterized in that part or all of the cytosines in the first mRNA or the second mRNA are chemically modified to improve the stability of the first mRNA or the second mRNA in vivo, and the chemical modification includes replacing at least 50% of the cytosines in the first mRNA or the second mRNA with 5-methylcytosine or 5-hydroxymethylcytosine.
The reprogramming factor anti-aging mRNA composition according to claim 4 is characterized in that the chemical modification includes replacing 100% of the uracil in the first mRNA or the second mRNA with N1-methylpseudouridine, pseudouracil or methyluracil, and replacing 100% of the cytosine in the first mRNA or the second mRNA with 5-methylcytosine or 5-hydroxymethylcytosine.
The reprogramming factor anti-aging mRNA composition according to claim 2, characterized in that the first mRNA comprises the following elements in sequence from 5' to 3' direction: a 5'UTR sequence, a signal peptide sequence, an Oct4 transcription factor coding sequence and a 3'UTR sequence;

Each of the second mRNAs includes the following elements in sequence from the 5’→3’ direction: a 5’UTR sequence, a target reprogramming factor coding sequence and a 3’UTR sequence, and each of the second mRNAs also includes a signal peptide sequence.
The reprogramming factor anti-aging mRNA composition according to any one of claims 1 to 6, characterized in that the reprogramming factor anti-aging mRNA composition further comprises:

a third mRNA encoding an RNA-dependent RNA polymerase;

Wherein, the molar amount of the third mRNA is 1 to 8 times the sum of the molar amounts of the first mRNA and the second mRNA.
The reprogramming factor anti-aging mRNA composition according to claim 7 is characterized in that the RNA-dependent RNA polymerase is a mutant replicase of the genus Alphavirus, and the mutant replicase produces a mutation at position 259 of the nsP2 region and a mutation at position 650 of the nsP2 region.
An anti-aging preparation, characterized in that it comprises a reprogramming factor anti-aging mRNA composition, sodium citrate dihydrate and sodium chloride, wherein the reprogramming factor anti-aging mRNA composition is the reprogramming factor anti-aging mRNA composition according to any one of claims 1 to 8, and the ratio of the sum of the masses of the first mRNA and the second mRNA in the reprogramming factor anti-aging mRNA composition, the mass of the sodium citrate dihydrate and the mass of the sodium chloride is 1 to 2:1 to 4:4 to 8.
A method for preparing the reprogramming factor anti-aging mRNA composition according to claim 1, characterized in that it comprises:

Synthesize the first mRNA;

synthesizing at least one second mRNA;

Part or all of the nucleotides in the first mRNA or the second mRNA are chemically modified to improve the stability of the first mRNA or the second mRNA in vivo, and the molar ratio of the first mRNA to the second mRNA is 2-8.
Use of the reprogramming factor anti-aging mRNA composition according to any one of claims 1 to 8 or the anti-aging preparation according to claim 9 in the preparation of a cell reediting reagent, in the preparation of a repair agent after organ damage, and in the preparation of an anti-aging drug.