Reprogramming factor preparation, biomaterial and use

Abstract

Provided are a reprogramming factor preparation, a biomaterial, and a use. The reprogramming factor preparation comprises a first expression system and a second expression system. The first expression system comprises a nucleic acid molecule encoding an Oct4 transcription factor, a nucleic acid molecule encoding a Sox2 transcription factor, and a nucleic acid molecule encoding a Klf4 transcription factor. The second expression system comprises a nucleic acid molecule encoding an Oct4 transcription factor, a nucleic acid molecule encoding a Glis1 transcription factor, and a nucleic acid molecule encoding a Lin28 transcription factor. The first expression system and the second expression system together form an OSKGL system (Oct4, Sox2, Klf4, Glis1 and Lin28). Said OSKGL system has a good epigenetic reprogramming effect in non-gene-edited living organisms such as wild-type mice, can achieve efficient expression of each reprogramming transcription factor, and has an anti-aging effect.

Description

Reprogramming factor preparations, biomaterials and uses Technical Field

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

[0002] Aging is a complex process characterized by chronic dysregulation of cellular function, leading to a gradual decline in tissue and organ function. While aging cannot be prevented, interventions aimed at restoring optimal function of these cellular processes can minimize its impact on lifespan and health in older adults.

[0003] Currently, using gene-edited mice to transiently express classic epigenetic reprogramming factors (i.e., Oct4, Sox2, and Klf4 transcription factors, OSK) can initiate partial epigenetic reprogramming, thereby reversing age-related pathological and pathophysiological changes in vitro and in vivo, improving organ function and extending the lifespan of aged mice. However, the expression of these OSK reprogramming factors in non-gene-edited living organisms, such as wild-type mice, is less effective.

[0004] Summary of the Invention

[0005] In view of the above problems, the embodiments of the present application provide a reprogramming factor preparation, biomaterial and use to solve the above technical problems.

[0006] In a first aspect, an embodiment of the present application provides a reprogramming factor preparation, comprising a first expression system and a second expression system, wherein the first expression system comprises a nucleic acid molecule encoding an Oct4 transcription factor, a nucleic acid molecule encoding a Sox2 transcription factor, and a nucleic acid molecule encoding a Klf4 transcription factor, and the second expression system comprises a nucleic acid molecule encoding an Oct4 transcription factor, a nucleic acid molecule encoding a Glis1 transcription factor, and a nucleic acid molecule encoding a Lin28 transcription factor.

[0007] Optionally, the first expression system and the second expression system are respectively adeno-associated virus AAV vectors.

[0008] Optionally, the first expression system comprises a nucleic acid molecule encoding an Oct4 transcription factor, a nucleic acid molecule encoding a P2A peptide, a nucleic acid molecule encoding a Sox2 transcription factor, a nucleic acid molecule encoding a T2A peptide, and a nucleic acid molecule encoding a Klf4 transcription factor.

[0009] Optionally, the nucleotide sequence of the first expression system is shown as SEQ ID NO.1.

[0010] Optionally, the second expression system comprises a nucleic acid molecule encoding an Oct4 transcription factor, a nucleic acid molecule encoding a P2A peptide, a nucleic acid molecule encoding a Glis1 transcription factor, a nucleic acid molecule encoding a T2A peptide, and a nucleic acid molecule encoding a Lin28 transcription factor.

[0011] Optionally, the nucleotide sequence of the second expression system is shown as SEQ ID NO.2.

[0012] Optionally, the first expression system and the second expression system are respectively a lentiviral expression vector or a Sendai virus expression vector or an expression vector containing a piggybac transposon.

[0013] In a second aspect, an embodiment of the present application provides a biomaterial, wherein the biomaterial is a host cell comprising the above-mentioned reprogramming factor preparation.

[0014] Optionally, when the first expression system and the second expression system are expressed in the host cell, the molar amount of the Oct4 transcription factor is twice the molar amount of the Sox2 transcription factor, the Klf4 transcription factor, the Glis1 transcription factor and the Lin28 transcription factor, respectively.

[0015] In a third aspect, the embodiments of the present application provide uses of the aforementioned reprogramming factor preparations or the aforementioned biomaterials in the preparation of drugs for improving organ function in elderly organisms or in the preparation of anti-aging drugs.

[0016] The reprogramming factor preparations, biomaterials and uses provided in the embodiments of the present application include a first expression system and a second expression system, wherein the first expression system includes a nucleic acid molecule encoding an Oct4 transcription factor, a nucleic acid molecule encoding a Sox2 transcription factor and a nucleic acid molecule encoding a Klf4 transcription factor, and the second expression system includes a nucleic acid molecule encoding an Oct4 transcription factor, a nucleic acid molecule encoding a Glis1 transcription factor and a nucleic acid molecule encoding a Lin28 transcription factor; through the above-mentioned method, the first expression system and the second expression system jointly form an OSKGL system (Oct4, Sox2, Klf4, Glis1 and Lin28), and the OSKGL system has a good epigenetic reprogramming effect in non-gene-edited living organisms such as wild-type mice, can achieve efficient expression of each reprogramming transcription factor, and has the effect of delaying aging.

[0017] These and other aspects of the present application will become more readily apparent from the description of the following embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] FIG1 shows a vector structure diagram of the first expression system in an embodiment of the present application.

[0019] FIG2 shows a vector structure diagram of the second expression system in an embodiment of the present application.

[0020] FIG3 shows a graph of the therapeutic effect evaluation results in Application Example 2 of the present application.

[0021] FIG4 shows a graph of the therapeutic effect evaluation results in Application Example 2 of the present application.

[0022] FIG5 shows the results of epigenetic age analysis in Application Example 2 of the present application.

[0023] FIG6 shows the results of epigenetic age analysis in Application Example 2 of the present application.

[0024] FIG7 shows a diagram of cardiac function evaluation results in Application Example 2 of the present application. DETAILED DESCRIPTION

[0025] 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 embodiments described 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 making creative efforts are within the scope of protection of this application.

[0026] References herein to "embodiments" mean that a particular feature, structure, or characteristic described in connection with the embodiments may be included in at least one embodiment of the present application. The appearance of this phrase in various places in the specification does not necessarily refer to the same embodiment, nor does it constitute an independent or alternative embodiment that is mutually exclusive of other embodiments. It is understood, both explicitly and implicitly, by those skilled in the art that the embodiments described herein may be combined with other embodiments.

[0027] The experimental methods in the following examples are conventional methods unless otherwise specified.

[0028] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0029] One embodiment of the present application provides a reprogramming factor preparation, which includes a first expression system and a second expression system, wherein the first expression system includes a nucleic acid molecule encoding an Oct4 transcription factor, a nucleic acid molecule encoding a Sox2 transcription factor, and a nucleic acid molecule encoding a Klf4 transcription factor, and the second expression system includes a nucleic acid molecule encoding an Oct4 transcription factor, a nucleic acid molecule encoding a Glis1 transcription factor, and a nucleic acid molecule encoding a Lin28 transcription factor.

[0030] In this embodiment, the first expression system and the second expression system together form the OSKGL system (Oct4, Sox2, Klf4, Glis1 and Lin28). The OSKGL system has a good epigenetic reprogramming effect in non-gene-edited living organisms such as wild-type mice, can achieve efficient expression of various reprogramming transcription factors, and has the effect of delaying aging.

[0031] In one embodiment, each of the aforementioned nucleic acid molecules may be DNA. The first expression system includes a DNA nucleic acid molecule encoding an Oct4 transcription factor, a DNA nucleic acid molecule encoding a Sox2 transcription factor, and a DNA nucleic acid molecule encoding a Klf4 transcription factor; the second system includes a DNA nucleic acid molecule encoding an Oct4 transcription factor, a DNA nucleic acid molecule encoding a Glis1 transcription factor, and a DNA nucleic acid molecule encoding a Lin28 transcription factor.

[0032] In one embodiment, each of the aforementioned nucleic acid molecules may be RNA. The first expression system includes an RNA nucleic acid molecule encoding an Oct4 transcription factor, an RNA nucleic acid molecule encoding a Sox2 transcription factor, and the second system includes an RNA nucleic acid molecule encoding an Oct4 transcription factor, an RNA nucleic acid molecule encoding a Klf4 transcription factor, an RNA nucleic acid molecule encoding a Glis1 transcription factor, and an RNA nucleic acid molecule encoding a Lin28 transcription factor.

[0033] In some embodiments, the first expression system and the second expression system are respectively adeno-associated virus (AAV) vectors.

[0034] In some embodiments, when the first expression system is an adeno-associated virus (AAV) vector, the DNA fragments of each transcription factor in the adeno-associated virus (AAV) vector can be connected with a 2A peptide. For example, the first expression system can sequentially include a nucleic acid molecule encoding an Oct4 transcription factor, a nucleic acid molecule encoding a P2A peptide, a nucleic acid molecule encoding a Sox2 transcription factor, a nucleic acid molecule encoding a T2A peptide, and a nucleic acid molecule encoding a Klf4 transcription factor.

[0035] Among them, 2A peptides are short peptides (generally 18 to 25 amino acids) derived from viruses. They are often called "self-cleaving" peptides and can produce multiple proteins from a single transcription product. 2A peptides are not completely "self-cleaving" but work by causing the ribosome to skip the synthesis of the glycine and proline peptide bonds at the C-terminus of the 2A element, ultimately leading to the separation of the 2A sequence end and the downstream product. The C-terminus of the upstream protein will have some additional 2A residues, while the N-terminus of the downstream protein will have an additional proline. P2A peptide, T2A peptide, E2A peptide, and F2A peptide are 2A peptide, P2A peptide, T2A peptide, E2A peptide, and F2A peptide, respectively, derived from four different viruses.

[0036] In some embodiments, the first expression system can be an adeno-associated virus (AAV) vector with ampicillin resistance.

[0037] In some embodiments, the nucleotide sequence of the first expression system is shown as SEQ ID NO.1.

[0038] In some embodiments, when the second expression system is an adeno-associated virus AAV vector, the DNA fragments of each transcription factor in the adeno-associated virus AAV vector can be connected with a 2A peptide. For example, the second expression system can include a nucleic acid molecule encoding an Oct4 transcription factor, a nucleic acid molecule encoding a P2A peptide, a nucleic acid molecule encoding a Glis1 transcription factor, a nucleic acid molecule encoding a T2A peptide, and a nucleic acid molecule encoding a Lin28 transcription factor.

[0039] In some embodiments, the nucleotide sequence of the second expression system is shown as SEQ ID NO.2.

[0040] In this embodiment, the first expression system can form an OSK system (Oct4, Sox2 and Klf4), and the second expression system can form an OGL system (Oct4, Glis1 and Lin28). When the OSKGL system (Oct4, Sox2, Klf4, Glis1 and Lin28) formed by the first expression system and the second expression system is expressed, the molar amount of the Oct4 transcription factor is approximately twice the molar amount of the Sox2 transcription factor, the Klf4 transcription factor, the Glis1 transcription factor and the Lin28 transcription factor, respectively. The preparation of this embodiment can be injected into the target organism, for example, by intravenous injection. After the preparation enters the target organism, it is transiently, periodically and systemically expressed in the target organism, thereby delaying the aging of the target organism. Exemplarily, the target organism can be a rodent (rats, mice), cats, dogs, bats, horses, rabbits, pigs, cattle, sheep, wolves, seals, finless porpoises, dolphins, whales, giraffes, Asian elephants, rhinos, non-human primates, humans, etc.

[0041] In some embodiments, the first expression system and the second expression system are respectively lentiviral expression vectors.

[0042] In some embodiments, the first expression system and the second expression system are respectively Sendai virus expression vectors.

[0043] In some embodiments, the first expression system and the second expression system are expression vectors comprising a piggybac transposon, respectively. For example, the first expression system and the second expression system can each be a recombinant vector having puromycin resistance, and the first expression system and the second expression system can be constructed according to the Gibson method and ligated using a Gibson enzyme. The vector sequence of the first expression system is characterized by inherent puromycin resistance and the ability to simultaneously express an Oct4 transcription factor, a Sox2 transcription factor, and a Klf4 transcription factor under the induction of doxycycline. The vector sequence of the second expression system is characterized by inherent puromycin resistance and the ability to simultaneously express an Oct4 transcription factor, a Glis1 transcription factor, and a lin28 transcription factor under the induction of doxycycline.

[0044] In this embodiment, the first expression system can form an OSK system (Oct4, Sox2 and Klf4), and the second expression system can form an OGL system (Oct4, Glis1 and Lin28). When the OSKGL system (Oct4, Sox2, Klf4, Glis1 and Lin28) formed by the first expression system and the second expression system is expressed, the molar amount of the Oct4 transcription factor is approximately twice the molar amount of the Sox2 transcription factor, the Klf4 transcription factor, the Glis1 transcription factor and the Lin28 transcription factor, respectively.

[0045] In Example 1 of the present application, the AAV9 viral vector for gene therapy is used as an example, but the first expression system and the second expression system in the preparation of the present application can also be not limited to other gene therapies such as non-viral vector transposons (Piggybac), plasmid DNA, nanolipid particles, cationic polymers, short peptide proteins, ligand-mediated targeting vectors, exosomes, etc., lentiviruses, retroviruses, adenoviruses, Sendai viruses, herpes simplex viruses, chemically modified mRNA, chemically modified mRNA and nanoparticles in gene therapy viral vectors, etc., and also include small molecules and combinations thereof that can enhance the efficiency of epigenetic reprogramming, such as Valproic acid, Chir-99021, E-616452, Tranylcypromine, Forskolin, TTNPB, Y-27632, ABT-869, Smoothened agonists, etc.

[0046] One embodiment of the present application provides a biomaterial, which is a host cell containing the above-mentioned reprogramming factor preparation.

[0047] As an embodiment, when the first expression system and the second expression system are expressed in host cells, the molar amount of Oct4 transcription factor is twice the molar amount of Sox2 transcription factor, Klf4 transcription factor, Glis1 transcription factor and Lin28 transcription factor, respectively.

[0048] As an embodiment, the host cell can be one of skin cells, cartilage cells, heart cells, ovarian cells, testicular cells or kidney cells, and the reprogramming factor preparation can partially reverse the aging of the host cells.

[0049] One embodiment of the present application provides the use of the above-mentioned reprogramming factor preparation or the above-mentioned biomaterial in the preparation of a drug for improving organ function in an elderly body or in the preparation of an anti-aging drug.

[0050] Example 1: Reprogramming Factor Preparation (AAV OSK+AAV OGL)

[0051] A first expression system was synthesized, wherein the first expression system was an adeno-associated virus AAV vector (hereinafter referred to as AAV OSK). The structure of the first expression system was shown in FIG1 , and the nucleotide sequence of the first expression system was shown in SEQ ID NO.1.

[0052] A second expression system was synthesized, wherein the second expression system was an adeno-associated virus AAV vector (hereinafter referred to as AAV OGL). The structure of the second expression system is shown in FIG2 , and the nucleotide sequence of the second expression system is shown in SEQ ID NO.2.

[0053] The first expression vector and the second expression vector were respectively assembled by the biological company SignaGen (https: / / signagen.com) to complete the AAV9 virus assembly to form the reprogramming factor preparation. In the reprogramming factor preparation, the first expression system (AAV OSK) was 1.86E13 vg / ml, and the second expression system (AAV9OGL) was 1.66E13 vg / ml.

[0054] Comparative Example 1: Reporter gene preparation (AAV GFP)

[0055] The reporter gene expression system that synthesizes green fluorescent protein (GFP) was assembled by the biological company SignaGen (https: / / signagen.com) using the AAV9 virus. The reporter gene expression system is an adeno-associated virus (AAV) vector that includes a DNA segment encoding green fluorescent protein.

[0056] Comparative Example 2: OSK expression system (AAV OSK)

[0057] The OSK expression system is the same as the first expression system. The structure is shown in Figure 1 and the nucleotide sequence is shown in SEQ ID NO. 1. The AAV9 virus was assembled by the biological company SignaGen (https: / / signagen.com).

[0058] Comparative Example 3: OGL expression system (AAV OGL)

[0059] The OGL expression system is the same as the second expression system. The structure is shown in Figure 2 and the nucleotide sequence is shown in SEQ ID NO. 2. The AAV9 virus was assembled by the biological company SignaGen (https: / / signagen.com).

[0060] Comparative Example 4: OSK+GL reprogramming factor preparation (AAV OSK+AAV GL)

[0061] A first expression system was synthesized, wherein the first expression system was an adeno-associated virus AAV vector (hereinafter referred to as AAV OSK). The structure of the first expression system was shown in FIG1 , and the nucleotide sequence of the first expression system was shown in SEQ ID NO.1.

[0062] A synthetic OL expression system is an adeno-associated virus (AAV) vector, comprising a nucleic acid molecule encoding a Glis1 transcription factor and a nucleic acid molecule encoding a Lin28 transcription factor. The difference between the OL expression system and the second expression system is that the OL expression system cannot express the Oct4 transcription factor.

[0063] The first expression system and OL expression system were respectively assembled by the biological company SignaGen (https: / / signagen.com) to complete the assembly of AAV9 virus.

[0064] Comparative Example 5: OSKM Expression System (AAV OSKM)

[0065] An OSKM expression system was synthesized, wherein the OSKM expression system was an adeno-associated virus (AAV) vector, and the OSKM expression system included a nucleic acid molecule encoding an Oct4 transcription factor, a nucleic acid molecule encoding a Sox2 transcription factor, a nucleic acid molecule encoding a Klf4 transcription factor, and a nucleic acid molecule encoding a C-MYC transcription factor.

[0066] Comparative Example 6: GL expression system (AAV GL)

[0067] A GL expression system was synthesized, wherein the GL expression system was an adeno-associated virus (AAV) vector, and the GL expression system included a nucleic acid molecule encoding a Glis1 transcription factor and a nucleic acid molecule encoding a Lin28 transcription factor.

[0068] Application Example 1

[0069] The preparation of Example 1, the expression system of Comparative Example 2, the OL expression system of Comparative Example 4, and the expression system of Comparative Example 5 were injected into wild mice, respectively, as shown in Table 1.

[0070] Table 1 Experimental group of application example 1

[0071] In Comparative Example 5, the expression of C-MYC transcription factor together with other transcription factors (Oct4 transcription factor, Sox2 transcription factor, Klf4 transcription factor) caused obvious toxicity and caused the death of mice. The transcription factor combinations in other experimental groups did not cause the death of mice.

[0072] Application Example 2

[0073] Wild mice were injected with the formulation of Example 1, the expression system of Comparative Example 1, the expression system of Comparative Example 2, the expression system of Comparative Example 3, and the formulation of Comparative Example 4, respectively, as shown in Table 2. Doxycycline was added to the mice's drinking water at a concentration of 2 mg / ml, and the drinking water contained 7.5% by mass of sucrose.

[0074] Table 2 Experimental group of application example 2

[0075] Efficacy evaluation:

[0076] The therapeutic effects of the preparation of Example 1, the expression system of Comparative Example 1, the expression system of Comparative Example 2, the expression system of Comparative Example 3, the preparation of Comparative Example 4, and Comparative Example 6 were evaluated respectively:

[0077] The survival rate of mice in each experimental group was calculated, and the Kaplan-Meier curve was drawn. Statistical analysis was performed using PRISM 9.0 software. P < 0.05 was marked with *, P < 0.01 was marked with **, P < 0.001 was marked with ***, and P < 0.0001 was marked with ****.

[0078] The results of efficacy evaluation are shown in Figures 3 and 4.

[0079] Epigenetic age analysis:

[0080] Epigenetic age analysis and methylation analysis were performed on the mice treated with the preparation of Example 1, the expression system of Comparative Example 1, the expression system of Comparative Example 2, the expression system of Comparative Example 3, the preparation of Comparative Example 4, and Comparative Example 6, respectively. The specific steps are as follows:

[0081] a. Sample preparation: Collect various corresponding tissues.

[0082] b. DNA extraction: Use a commercial DNA extraction kit to extract DNA from the samples.

[0083] c. Methylase treatment: DNA is treated with a commercial methylase reagent to distinguish methylated cytosine from unmethylated cytosine.

[0084] d. Methylation detection: Sequencing was performed using the above-mentioned WGBS sequencing method.

[0085] e. Data Analysis: Data were input into the bioinformatics software R package. Data analysis included quality control, data cleaning, data preprocessing, feature extraction, and statistical analysis.

[0086] f. Calculation of aging rate.

[0087] g. Input the above methylation data into the online epigenetic age calculator (https: / / dnamage.genetics.ucla.edu / home).

[0088] h. Select Horvath clock and Hannum clock.

[0089] i. After running the analysis, the epigenetic age is generated.

[0090] j. Calculation of aging rate: The apparent age / calendar age of the mouse determined by the above method is the aging rate of the body, tissues, and organs.

[0091] The results of epigenetic age analysis are shown in Figures 5 and 6.

[0092] Cardiac function assessment:

[0093] Cardiac ultrasound was performed on the mice treated with the preparation of Example 1, the expression system of Comparative Example 1, the expression system of Comparative Example 2, the expression system of Comparative Example 3, the preparation of Comparative Example 4, and Comparative Example 6 to evaluate the cardiac contractile function:

[0094] A Vevo 3100 high-frequency ultrasound instrument (Fujifilm, VisualSonics) was used to assess cardiac function. Inhalation anesthesia (2% isoflurane) was maintained during the examination. The mouse body temperature was maintained at 37 degrees Celsius, the heart rate was maintained at >450 beats / min, and the ejection fraction (EF, %) was calculated using the Vevolab Imaging Software (Fujifilm, VisualSonics).

[0095] The cardiac assessment results are shown in Figure 7.

[0096] Experimental results and analysis

[0097] In 120-week-old mice, doxycycline was used systemically to induce AAV to express various transcription factors or green fluorescent protein. The expression cycle was doxycycline-induced expression for 1 week and no expression for 1 week, and so on. As shown in Figures 3 and 4, the reporter gene preparation (AAV GFP, Comparative Example 1) extended the lifespan of 120-year-old mice by an average of 12.24 months; the gene cocktail therapy of the OSK expression system (AAV OSK, Comparative Example 2) can significantly extend the lifespan of 120-week-old mice, with an average increase of 16.32 months; and the gene cocktail therapy of the reprogramming factor preparation (AAV OSK+AAV OGL, Example 1) can significantly extend the lifespan of 120-week-old mice by 25.38 months, which is significantly improved compared with the average increase of 16.32 months of lifespan of the OSK expression system (AAV OSK, Comparative Example 2). This confirms that the gene cocktail therapy of the reprogramming factor preparation (AAV OSK+AAV OGL, Example 1) is more efficient than the gene cocktail therapy of the OSK expression system (AAV OSK, Comparative Example 2). Compared with the reprogramming factor preparation (AAV OSK+AAV OGL, Example 1) of the present application and the OSK+GL reprogramming factor preparation (AAV OSK+AAV GL, Comparative Example 4), the preparation of Example 1 can more significantly extend the lifespan of mice at 120 weeks of age, confirming that when the expression level of Oct4 transcription factor is about 2 times the expression level of other transcription factors, it is the most efficient and optimized reprogramming factor combination, while there is no significant difference in the effect of Example 1 and Comparative Example 2 on extending the lifespan of mice at 120 weeks of age. There is no significant difference in the effects of Example 1, Comparative Example 6, and Comparative Example 1, which also confirms that when the expression level of Oct4 transcription factor is about 2 times the expression level of other transcription factors, it is the most efficient and optimized reprogramming factor combination.

[0098] In 120-week-old mice, doxycycline was used systemically to induce AAV to express various transcription factors or green fluorescent protein. The expression cycle was doxycycline-induced expression for 1 week and no expression for 1 week. After 10 cycles, please refer to Figures 5 and 6. Compared with the reporter gene preparation (AAV GFP, Comparative Example 1) gene cocktail therapy, the OSK expression system (AAV OSK, Comparative Example 2) gene cocktail therapy significantly reduced the epigenetic age of the liver and heart of the elderly organisms; and compared with the OSK expression system (AAV OSK, Comparative Example 2) gene cocktail therapy, the reprogramming factor preparation (AAV OSK+AAV OGL, Example 1) gene cocktail therapy further significantly reduced the epigenetic age of the liver and heart of the elderly organisms. Moreover, compared with the OSK+GL reprogramming factor preparation (AAV OSK+AAV GL, Comparative Example 4), the reprogramming factor preparation (AAV OSK+AAV OGL, Example 1) can more significantly reduce the epigenetic age of the liver and heart, confirming that when the Oct4 transcription factor expression level is twice the expression level of other transcription factors, it is the most efficient and optimized reprogramming factor combination; while there is no significant difference between Comparative Example 4 and Comparative Example 2 in the effect of reducing the epigenetic age of the liver and heart, and there is no significant difference between Comparative Example 4, Comparative Example 6 and Comparative Example 1, which also confirms that the OSKGL combination and the Oct4 transcription factor expression level is twice the expression level of other transcription factors are the most efficient and optimized reprogramming factor combination.

[0099] Please refer to Figure 7. Compared with the reporter gene preparation (AAV GFP, Comparative Example 1) gene cocktail therapy, the OSK expression system (AAV OSK, Comparative Example 2) gene cocktail therapy cannot significantly improve the ejection fraction (EF%) of the elderly heart function indicator; while compared with the OSK expression system (AAV OSK, Comparative Example 2) gene cocktail therapy, the reprogramming factor preparation (AAV OSK+AAV OGL, Example 1) gene cocktail therapy can significantly improve the ejection fraction (EF%) of the elderly heart function. Moreover, compared with the OSK+GL reprogramming factor preparation (AAV OSK+AAV GL, Comparative Example 4), the reprogramming factor preparation (AAV OSK+AAV OGL, Example 1) can more significantly improve the cardiac function of the elderly body, confirming that when the Oct4 transcription factor expression level is twice that of other transcription factors, it is the most efficient and optimized reprogramming factor combination; while there is no significant difference between Comparative Example 4 and Comparative Example 2 in the effect of improving the lifespan of the elderly body, and there is no significant difference in the effects of Comparative Example 4, Comparative Example 6 and Comparative Example 1, which also confirms that the OSKGL combination and the Oct4 transcription factor expression level is twice that of other transcription factors are the most efficient and optimized reprogramming factor combination.

[0100] The above is only an implementation method of the present application. It should be pointed out that for ordinary technicians in this field, improvements can be made without departing from the creative concept of the present application, but these all fall within the scope of protection of the present application.

Claims

1. A reprogramming factor preparation, characterized in that: The invention comprises a first expression system and a second expression system, wherein the first expression system comprises a nucleic acid molecule encoding an Oct4 transcription factor, a nucleic acid molecule encoding a Sox2 transcription factor and a nucleic acid molecule encoding a Klf4 transcription factor, and the second expression system comprises a nucleic acid molecule encoding an Oct4 transcription factor, a nucleic acid molecule encoding a Glis1 transcription factor and a nucleic acid molecule encoding a Lin28 transcription factor.

2. The reprogramming factor preparation according to claim 1, characterized in that The first expression system and the second expression system are respectively adeno-associated virus AAV vectors.

3. The reprogramming factor preparation according to claim 2, characterized in that The first expression system includes a nucleic acid molecule encoding an Oct4 transcription factor, a nucleic acid molecule encoding a P2A peptide, a nucleic acid molecule encoding a Sox2 transcription factor, a nucleic acid molecule encoding a T2A peptide, and a nucleic acid molecule encoding a Klf4 transcription factor.

4. The reprogramming factor preparation according to claim 3, characterized in that The nucleotide sequence of the first expression system is shown in SEQ ID NO.

1.

5. The reprogramming factor preparation according to claim 2, characterized in that The second expression system comprises a nucleic acid molecule encoding an Oct4 transcription factor, a nucleic acid molecule encoding a P2A peptide, a nucleic acid molecule encoding a Glis1 transcription factor, a nucleic acid molecule encoding a T2A peptide, and a nucleic acid molecule encoding a Lin28 transcription factor.

6. The reprogramming factor preparation according to claim 5, characterized in that The nucleotide sequence of the second expression system is shown as SEQ ID NO.

2.

7. The reprogramming factor preparation according to claim 1, characterized in that The first expression system and the second expression system are respectively a lentiviral expression vector or a Sendai virus expression vector or an expression vector containing a piggybac transposon.

8. A biomaterial, characterized in that The biological material is a host cell comprising the reprogramming factor preparation according to any one of claims 1 to 7.

9. The biomaterial according to claim 8, characterized in that When the first expression system and the second expression system are expressed in the host cell, the molar amount of the Oct4 transcription factor is twice the molar amount of the Sox2 transcription factor, the Klf4 transcription factor, the Glis1 transcription factor, and the Lin28 transcription factor, respectively.

10. Use of the reprogramming factor preparation according to any one of claims 1 to 7 or the biomaterial according to any one of claims 8 to 9 in the preparation of drugs for improving organ function in the elderly or in the preparation of anti-aging drugs.

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