Inactivated telomerase, adenovirus and artificial mRNA containing the same, and uses thereof

Abstract

The present invention provides an inactivated telomerase, which is a double-mutant telomerase with double mutations Y707F and D868A, respectively. The present invention also provides adenovirus-inactivated telomerase and artificial mRNA-inactivated telomerase with double mutations, as well as their use in treating diseases associated with telomere shortening in non-dividing cells. The present invention can inhibit the activity of telomerase TERT in non-dividing cells to form inactivated telomerase CI-TERT, while simultaneously restricting telomerase from leaving the cell nucleus. This combination retains telomerase in the cell nucleus, protecting telomeres from shortening and preventing cellular carcinogenesis caused by telomere lengthening by telomerase. While reducing the risk of carcinogenesis, telomerase can be retained in the cell nucleus to protect telomere ends, thereby suppressing the progression of diseases caused by telomere shortening.

Description

[Technical field]

[0001] The present invention relates to the fields of genetic engineering and cytology technology, specifically to inactivated telomerase and uses thereof. [Background technology]

[0002] Telomere is a complex of multiple repeat non-transcribed DNA (Deoxyribonucleic Acid) and protein at the end of human eukaryotic chromosome. Research has revealed that telomeres have a certain relationship with aging, cell division control, and disease. Telomerase is an enzyme that plays a role in extending telomeres in cells. It is a basic nuclear protein reverse transcriptase, which can add telomeric DNA to the end of eukaryotic chromosomes, replenish telomeres worn down by DNA replication, repair and extend telomeres, and prevent telomeres from being worn down by cell division.

[0003] Over the past few decades, gene therapy has gradually turned from a scientist's dream into reality. Gene therapy vectors have been the key to the whole of gene therapy so far. At present, AAV (Adenovirus Associated Virus) has been widely studied as a vector for gene therapy, since it can be integrated into human chromosomes by transfection. However, adenovirus AAV has low specific recognition for cells in the human body, and the virus has the risk of inducing immune storm. Research has revealed that telomere shortening is closely related to various diseases and aging, and telomere lengthening can alleviate diseases or have a certain therapeutic effect. At present, in the laboratory, adenovirus-telomerase AAV-TERT is directly injected into the test body to overexpress active telomerase, thereby achieving the purpose of telomere lengthening. However, in such a non-specific design, cells with mutations cannot be prevented from turning into cancer cells by active telomerase. Therefore, the direction that those skilled in the art should research is how to use appropriate genes and their vectors to retain telomerase in the cell nucleus to protect the telomere ends while reducing the risk of cancer and suppressing the worsening of diseases caused by telomere shortening. Summary of the Invention

[0004] In order to overcome the deficiencies and shortcomings in the prior art, the present invention aims to provide an inactivated telomerase, an adenovirus-inactivated telomerase having the same, and an artificial mRNA-inactivated telomerase, and uses thereof.

[0005] To achieve the above object, according to a first aspect of the present invention, there is provided an inactivated telomerase, which is a double mutant telomerase, the double mutation points of which are Y707F and D868A, respectively.

[0006] According to a second aspect of the present invention, there is provided an adenovirus-inactivated telomerase comprising a double mutant telomerase, wherein the double mutation points of said double mutant telomerase are Y707F and D868A, respectively.

[0007] According to a third aspect of the present invention, there is provided an artificial mRNA-inactivated telomerase comprising a double mutant telomerase, the double mutation points of said double mutant telomerase being Y707F and D868A, respectively.

[0008] According to a fourth aspect of the present invention, there is provided the use of said adenovirus-inactivated telomerase or said artificial mRNA-inactivated telomerase for the treatment of a disease associated with telomere shortening in non-dividing cells.

[0009] Additionally, non-dividing cells include cardiac muscle cells, skeletal muscle cells and neuronal cells.

[0010] Furthermore, diseases associated with telomere shortening in non-dividing cells preferably include diseases associated with telomere shortening in cardiomyocytes.

[0011] Furthermore, diseases associated with telomere shortening in cardiomyocytes include dilated cardiomyopathy (DCM), hypertrophic cardiomyopathy (HCM), laminopathies and rare telomere shortening heart diseases.

[0012] Furthermore, rare telomere shortening heart diseases include Duchenne muscular dystrophy (DMD).

[0013] According to the above technical solution, the present invention provides a double mutant inactive telomerase (CI-TERT, Catalytically Inactive TERT) with mutation points Y707F and D868A, respectively, in which the mutation point Y707F keeps telomerase in the cell nucleus and restricts its release, so that telomerase is long-term engaged with the telomere end to form a protective structure. The mutation point D868A inactivates telomerase, which can prevent the uncontrollable elongation of telomeres and the possible induction of cancer due to the inactivation of telomerase. In order to realize the function of double mutation inactivated telomerase, the inventor found that adenovirus and artificial mRNA can be used as a vector for double mutation inactivated telomerase, and double mutation inactivated telomerase can be generated in the recipient by injecting adenovirus-inactivated telomerase and / or artificial mRNA-inactivated telomerase into the recipient; that is, the technical solution of the present invention generates double mutation inactivated telomerase in the recipient cell, and further overexpresses inactivated telomerase CI-TERT, so that the double mutation inactivated telomerase can exert the corresponding function through the cooperation of double mutation points, which not only reduces the risk of cell canceration, but also keeps telomerase in the cell nucleus to protect telomere ends, and inhibits the aggravation of diseases caused by telomere shortening.

[0014] The present invention has the following beneficial effects over the prior art: 1. Preventing telomere shortening and preventing and treating various diseases and aging associated with telomere shortening, particularly telomere shortening of non-dividing cells. 2. It can lengthen the telomeres of various diseases and senescent cells associated with telomere shortening, especially telomere shortening of non-dividing cells, and has a certain therapeutic effect on alleviating diseases. 3. It can suppress cell death caused by telomere shortening. 4. It can protect the ends of telomeres and prevent the risk of cell cancer caused by excessive telomere growth. [Brief description of the drawings]

[0015] The drawings constituting a part of the present invention are provided for a further understanding of the present invention, and the exemplary embodiments of the present invention and the description thereof are used to interpret the present invention, and are not intended to unduly limit the present invention. [Figure 1] FIG. 1 shows a flow chart of the working principle of the present invention. [Diagram 2] FIG. 2 shows a schematic diagram of localizing telomeres (telomeric repeat sequence staining TelC) and measuring their length by QFISH in healthy cardiomyocytes (cardiac sarcomere staining cTnT). [Diagram 3] FIG. 3 shows a schematic diagram of localizing telomeres (telomeric repeat staining TelC) and measuring their length by QFISH in diseased cardiomyocytes (cardiac sarcomere staining cTnT). [Figure 4] FIG. 4 shows colocalized immunofluorescence staining of telomere ends (telomeric repeat stain TelC) and nucleic acid damage (nucleic acid damage signal stain 53BP1) in diseased cardiomyocytes (myocardial sarcomere stain cTnT). [Diagram 5] FIG. 5 shows co-localized immunofluorescence staining of telomere ends (telomeric repeat staining TelC) and telomerase (telomerase TERT) in diseased cardiomyocytes. [Figure 6] FIG. 6 shows a diagram of telomerase activity detection (TRAP Assay). [Figure 7] FIG. 7 shows a detection diagram of a nucleic acid-protein colocalization (ChIP-qPCR) test. [Figure 8] FIG. 8 shows a schematic diagram comparing the telomere length of artificial mRNA-telomerase (mRNA-TERT) and artificial mRNA-inactivated telomerase (mRNA-CI-TERT) in human-derived cardiomyocytes. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0016] The present invention will be described in more detail below with reference to the drawings through specific embodiments and examples. In addition, the embodiments and requirements of the embodiments of the present invention may be combined with each other unless there is a contradiction. The present invention will be described in more detail below with reference to the drawings in combination with the embodiments.

[0017] According to one representative embodiment of the present invention, there is provided an inactivated telomerase, which is a double mutant telomerase, the double mutation points being Y707F and D868A, respectively. Figure 1 shows a flow chart of the working principle of the present invention.

[0018] According to the above technical solution, the mutation of the mutation point Y707F keeps telomerase in the cell nucleus, restricts the release of telomerase, and engages telomerase with the telomere end in the long term to form a protective structure. The mutation of the mutation point D868A inactivates telomerase, preventing the inactivation of telomerase from causing uncontrollable telomere elongation and the possibility of inducing cancer. The double mutant inactive telomerase CI-TERT with the mutation points Y707F and D868A provided by the present invention can inhibit the activity of telomerase TERT, making it inactive telomerase CI-TERT, while inhibiting the release of telomerase from the cell nucleus. The double mutation sites Y707F and D868A work together to retain telomerase in the cell nucleus to hold the telomere end, and telomerase can be engaged with the telomere end for a long period of time to form a protective structure, while preventing the loss of control of telomere elongation caused by telomerase detachment and the resulting cell cancerization. Therefore, the double mutation inactivated telomerase CI-TERT provided by the present invention can retain telomerase in the cell nucleus to protect the telomere end while reducing the risk of cancerization, and suppress the aggravation of diseases caused by telomere shortening.

[0019] According to one representative embodiment of the present invention, there is provided an adenovirus-inactivated telomerase AAV-CI-TERT, which contains a double mutant telomerase, and the double mutation points are Y707F and D868A, respectively. The adenovirus-inactivated telomerase AAV-CI-TERT of this embodiment is: Step S1 of culturing the double mutant inactivated telomerase CI-TERT plasmid, the above-mentioned mutation points of which are Y707F and D868A, respectively, using bacteria; Step S2 of transforming the double mutant inactivated telomerase CI-TERT plasmid into adenovirus-293T or AAV-293T cells using lipofectamin and culturing the cells; Step S3, after 7 days of culture, extracting the adenovirus or AAV virus carrying the double mutant inactivated telomerase CI-TERT plasmid by centrifugation; It is made by:

[0020] This resulted in a double mutant adenovirus-inactivated telomerase CI-TERT or AAV virus-inactivated telomerase CI-TERT, with the mutation points Y707F and D868A, respectively.

[0021] The extracted adenovirus carrying the double mutant inactivated telomerase CI-TERT plasmid or the AAV adeno-associated virus carrying the double mutant inactivated telomerase CI-TERT plasmid is added to the medium (cell culture) and cultured for 24 to 48 hours, after which the culture medium is replaced or injected into the recipient, and it is observed whether the recipient itself has produced adenovirus-inactivated telomerase or AAV-CI-TERT.

[0022] In each of the above steps, the successful transformation of the double mutant inactivated telomerase CI-TERT plasmid can be verified by gene detection. After injection or infusion into the recipient, immunofluorescence staining can also be performed in the animal recipient or biological sample to verify that the expected effect has been achieved.

[0023] Specifically, the immunofluorescence staining includes the following steps. 1. Sample Staining The sample was then marked with a cut-off pen around the sample, and 10 micrograms per milliliter (μg / mL) of telomere peptide nucleic acid PNA antibody was added to perform TelC telomere repeat sequence staining, and the slide was placed in a staining box to prevent light exposure. The sample was covered with a cover glass and placed in an oven at 84°C for 7 minutes. The cover glass was removed and washed twice with PNA washing solution (15 minutes each time), and after washing, it was further washed twice with PBST (5 minutes each time). After washing, it was further washed for 1 hour with a blocking solution prepared by adding phosphate-buffered saline to 10% calf serum at room temperature, and then stained for 2 hours with a cardiac sarcomere (cTnT) primary antibody diluted 1000 times at room temperature. The sample was again washed three times with blocking solution (5 minutes each time), and then stained for 1 hour with an anti-mouse 488alexa488 secondary antibody diluted 1:1000. The samples were washed twice with peptide nucleic acid PNA washing solution and PBST (15 min each time), stained for 5 min with DAPI, a cell nucleus stain, in molecular-free pure water at a total concentration of 1 μg / mL, washed three times with phosphate-buffered saline (5 min each time), mounted with mounting medium, and stored at 4°C until photographed under a microscope.

[0024] 2. Measuring telomere length Using Telometer, an ImageJ plug-in developed by the laboratory of Professor Alan Meeker at Hopkins University in the United States, cell nuclei were selected in the photographs of the stained samples, and telomere length was measured by comparing it with the fluorescence intensity of DAPI in the cell nuclei using TelC.

[0025] According to the above technical solution, adenovirus carrying inactivated telomerase CI-TERT plasmid or AAV adeno-associated virus carrying inactivated telomerase CI-TERT plasmid is injected into the recipient, and double mutant inactivated telomerase CI-TERT is generated in the recipient, and the double mutant points work together, where the Y707F mutant point keeps telomerase in the cell nucleus, restricts telomerase from being released, and allows telomerase to be engaged with the telomere end in the long term to form a protective structure. The D868A mutant point inactivates telomerase, and the inactivation of telomerase prevents the uncontrollable elongation of telomeres and the occurrence of malignant tumors. In the technical solution of the present invention, adenovirus is used as a vector to successfully inject double mutant inactivated telomerase into the recipient, and double mutant inactivated telomerase CI-TERT is generated in the recipient, which reduces the risk of canceration while keeping telomerase in the cell nucleus to protect the telomere end, thereby suppressing the aggravation of diseases caused by telomere shortening.

[0026] According to one representative embodiment of the present invention, there is provided an artificial mRNA-silencing telomerase, which comprises double mutant telomerase, and the double mutation points of the double mutant telomerase are Y707F and D868A respectively. The preparation of the artificial mRNA-silencing telomerase DNA nucleic acid sequence is as follows: Artificial mRNA is also called mmRNA, and artificial mRNA is artificial messenger RNA, and the artificial mRNA-inactivated telomerase or mmRNA-CI-TERT used in this embodiment is a double mutant artificial mRNA-inactivated telomerase or mmRNA-CI-TERT with Y707F mutation (retained in cell nucleus) and D868A mutation (no activity). The double mutant artificial mRNA-inactivated telomerase or mmRNA-CI-TERT with mutation points of Y707F and D868A respectively is injected into the recipient, and it is observed whether the recipient itself produces artificial mRNA-inactivated telomerase or mmRNA-CI-TERT.

[0027] In each step above, the successful transformation of the double mutant inactivated telomerase CI-TERT plasmid can be verified by gene detection. After injection or infusion into the recipient, immunofluorescence staining can also be performed on the animal recipient or biological sample to verify that the expected effect has been achieved.

[0028] Specifically, the immunofluorescence staining includes the following steps. 1. Sample staining: Draw around the sample with a cut-off pen, add 10 micrograms / milliliter (μg / mL) telomere peptide nucleic acid PNA antibody to perform TelC telomere repeat sequence staining, and place the slide in a staining box to prevent light exposure. Cover the sample with a cover glass and leave it in an oven at 84°C for 7 minutes. Remove the cover glass and wash twice with PNA washing solution (15 minutes / time), and after washing, wash twice more with PBST (5 minutes / time). After washing, wash for 1 hour with blocking solution prepared by adding phosphate-buffered saline to 10% calf serum at room temperature, and then stain with cardiac sarcomere (cTnT) primary antibody diluted 1000 times at room temperature for 2 hours. Again, wash the sample with blocking solution three times (5 minutes / time), and then stain with anti-mouse 488alexa488 secondary antibody diluted 1:1000 for 1 hour. The sections were washed twice with peptide nucleic acid PNA washing solution and PBST (15 min each time), stained for 5 min with the cell nucleus stain DAPI at a total concentration of 1 μg / mL in molecular-free pure water, washed three times with phosphate-buffered saline (5 min each time), mounted with mounting medium, and stored at 4°C until imaging. 2. Measurement of telomere length: Using the ImageJ plug-in Telometer (a software for measuring the length of telomere ends) developed by the laboratory of Professor Alan Meeker at Hopkins University in the United States, telomere length was measured by comparing the fluorescence intensity of DAPI in the cell nuclei by TelC with the cell nuclei in photographs of stained samples.

[0029] According to the above technical solution, the artificial mRNA and / or mmRNA with the double mutation inactivated telomerase CI-TERT plasmid, whose mutation points are Y707F and D868A respectively, is injected into the recipient to generate the double mutation inactivated telomerase CI-TERT in the recipient, and the double mutation points work together, where the mutation point Y707F keeps telomerase in the cell nucleus, restricts the telomerase from being released, and prevents telomeres from shortening. The mutation point D868A is mutated to inactivate telomerase, which prevents the uncontrollable elongation of telomeres and the possible induction of cancer due to the inactivation of telomerase. In the technical solution of the present invention, the artificial mRNA or mmRNA is used as a vector to successfully inject the double mutation inactivated telomerase into the recipient to generate the double mutation inactivated telomerase CI-TERT in the recipient, which keeps telomerase in the cell nucleus to protect the telomere ends while reducing the risk of canceration, and inhibits the aggravation of diseases caused by telomere shortening.

[0030] According to one representative embodiment of the present invention, there is provided a use of the adenovirus-inactivated telomerase in the above embodiment or the artificial mRNA-inactivated telomerase in the above embodiment for treating a disease associated with telomere shortening in non-dividing cells.

[0031] It should be noted that telomere shortening in non-dividing cells can cause aging, cell division control, and other diseases. According to the technical solution of the present invention, adenovirus-inactivated telomerase AAV-CI-TERT or artificial mRNA-inactivated telomerase mmRNA-CI-TERT generates double-mutated inactivated telomerase in the receptor, and the mutation point Y707F keeps the telomerase receptor in the cell nucleus, restricts the release of telomerase, and engages telomerase with the telomere end in the long term to form a protective structure. The mutation point D868A is mutated to inactivate telomerase, which prevents the uncontrollable elongation of telomeres and the possible induction of cancer due to the inactivation of telomerase. Therefore, the technical solution of the present invention successfully generates a double mutation inactivated telomerase CI-TERT in the receptor, effectively controlling the expression of telomerase and the type of receptor cells, reducing the risk of canceration, while overexpressing active telomerase, so that telomerase is retained in the cell nucleus to protect the telomere ends, preventing telomere shortening, and preventing cell death and disease aggravation caused by telomere shortening in non-dividing cells, thereby preventing and treating various diseases and aging associated with telomere shortening, and can lengthen the telomeres of various diseases and aging cells associated with telomere shortening, achieving therapeutic effects on alleviated diseases.

[0032] Preferably, non-dividing cells include cardiac muscle cells, skeletal muscle cells and nerve cells. Non-dividing cells include, but are not limited to, cardiac muscle cells, skeletal muscle cells and nerve cells, etc. Cardiomyocytes, skeletal muscle cells and nerve cells, etc., all of which suffer from telomere shortening or gradual shortening, leading to abnormalities and diseases, and the double mutant telomerase can effectively control the expression of telomerase and the type of receptor cells in non-dividing cells to achieve the corresponding functions.

[0033] Preferably, diseases associated with telomere shortening in non-dividing cells include diseases associated with telomere shortening in cardiomyocytes.

[0034] Preferably, the diseases associated with telomere shortening in cardiomyocytes include dilated cardiomyopathy (DCM), hypertrophic cardiomyopathy (HCM), laminopathies and rare telomere shortening heart diseases.

[0035] More preferably, the rare telomere shortening heart disease comprises Duchenne muscular dystrophy DMD.

[0036] Research has revealed that telomere shortening in cardiomyocytes is the cause of rare heart diseases, such as Duchenne muscular dystrophy (DMD), and death. Diseases associated with telomere shortening in cardiomyocytes further include laminopathy, most dilated cardiomyopathy (DCM) in Titinopathy, and hypertrophic cardiomyopathy (HCM). The inventors have found that diseases associated with telomere shortening in cardiomyocytes are manifested as gradual telomere shortening. Diseases caused by telomere shortening are not limited to genetic cardiomyopathy, and research has revealed that telomere shortening is associated with a series of diseases. According to the technical solution of the present invention, a double mutant inactivated telomerase with mutation points of Y707F and D868A, respectively, is injected into the cardiomyocytes of the recipient using adenovirus or artificial mRNA as a vector, and a double mutant inactivated telomerase with Y707F and D868A is successfully generated in the cardiomyocytes of the recipient. Mutation of the mutation point Y707F keeps telomerase in the cell nucleus and restricts the release of telomerase, thereby preventing telomere shortening. Mutation of the mutation point D868A inactivates telomerase, and the inactivation of telomerase prevents uncontrollable telomere growth. For the abnormality of gradual telomere shortening among cardiomyocyte abnormalities, the double mutant inactivated telomerase of the present invention can improve the continuous deterioration of the condition caused by gradual telomerase shortening, and at the same time, it can also prevent the occurrence of telomere shortening by intervention or treatment at a sufficiently early stage. After a large number of tests, the inventors further found that telomere shortening due to heart failure causes nucleic acid damage, reduces the function and number of mitochondria, and further affects myocardial contractile function. By protecting the telomere ends with inactivated telomerase CI-TERT, it is possible to suppress the deterioration of the disease and prevent nucleic acid damage, reduction in the function and number of mitochondria, and deterioration of myocardial function.

[0037] The beneficial effects of the present invention will now be further illustrated with reference to the following examples.

[0038] Working Example: According to one exemplary embodiment of the present invention, adenovirus-inactivated telomerase AAV-CI-TERT is used to treat heart disease.

[0039] When the length of the telomere ends in the heart slices of the patients with cardiomyopathy was measured by localization using immunofluorescence staining, it was found that the patients with cardiomyopathy had abnormal telomere shortening, which gradually caused cell death and led to the deterioration of the disease and heart failure. Of note, telomere shortening was not detected in other biological samples from the patients. Tests have shown that this phenomenon can be observed in cardiomyocytes differentiated from human-derived stem cells as well, and the telomere length in the nuclei of cardiomyocytes differentiated from human-derived stem cells can be measured by localization using immunofluorescence staining, proving that cardiomyocytes differentiated from human-derived stem cells can be used as a test model for abnormal telomere shortening in heart disease, as shown in Figures 2 and 3. At the level of human-derived stem cells, there is no difference in telomere length between healthy cells and diseased cells. Compared to cardiomyocytes differentiated from healthy human-derived stem cells, cardiomyocytes differentiated from diseased human-derived stem cells showed telomere shortening. This means that abnormal telomere shortening occurs when cardiomyocytes are not dividing, and under normal circumstances, telomere shortening occurs during cell division, and cells with too short telomeres are removed during division. The inventors have repeatedly tested and found that the abnormal telomere shortening occurs because reactive oxygen species (ROS) in the progression of the disease activate the mitochondrial protective function of telomerase, causing telomerase to leave the cell nucleus and the telomere ends to lose protection. The inventors have further found that this abnormal telomere shortening is not an acute shortening, but a gradual and gradual shortening. This means that telomere shortening can be effectively suppressed or prevented by intervention or treatment at an early stage of the disease.

[0040] Table 1 shows telomere length measurements of patient myocardial samples.

[0041] Table 1 demonstrates abnormal telomere shortening in non-dividing cells (cardiomyocytes) in patients with cardiomyopathy.

[0042] [Table 1]

[0043] Table 2 shows telomere length measurements in smooth muscle sections from patients, demonstrating that abnormal telomere shortening in patients with cardiomyopathy is present only in cardiac myocytes.

[0044] [Table 2]

[0045] Table 3 shows telomere length measurements of human-derived stem cells and cardiomyocytes differentiated from human-derived stem cells. Abnormal telomere shortening has been demonstrated to occur in non-dividing diseased cardiomyocytes.

[0046] [Table 3]

[0047] Follow-up studies of telomere length are shown in Table 4. This abnormal telomere shortening was not acute, but rather demonstrated gradual and gradual shortening.

[0048] [Table 4] TIFF0007599553000005.tif27153

[0049] Further comparison of cardiomyocytes differentiated from healthy human-derived stem cells with cardiomyocytes differentiated from human-derived stem cells from disease patients revealed that the diseased cells exhibited higher nucleic acid damage signal 53BP1 at the telomere ends in the nucleus, and the number of mitochondria was obviously reduced, as shown in Figure 4, and the cellular contractile function, i.e., the contractile force, was reduced.

[0050] Table 5 shows the measurement of nucleic acid damage signals in cardiomyocytes, demonstrating that there are more nucleic acid damage signals in diseased cardiomyocytes.

[0051] [Table 5]

[0052] Table 6 shows the measurement of mitochondrial number in cardiomyocytes, demonstrating a clear decrease in mitochondrial number in diseased cells.

[0053] [Table 6]

[0054] Table 7 shows the measurement of the contractile force of cardiomyocytes, which has been proven to affect and reduce the function of diseased cells. The unit is nano-newton, abbreviated as nN.

[0055] [Table 7]

[0056] In order to suppress telomere shortening and avoid excessive elongation of telomere ends, the embodiment of the present invention created a human telomerase sequence with two mutation points, which was named inactivated telomerase or CI-TERT. One of the two mutation points of inactivated telomerase or CI-TERT is the mutation point Y707F, which keeps telomerase in the cell nucleus, and the other is the mutation point D868A, which inactivates telomerase. Inactivated telomerase was injected into the recipient by adenovirus (AAV) or artificial mRNA (mmRNA), that is, adenovirus-inactivated telomerase or artificial mRNA-inactivated telomerase was injected into the recipient. As shown in FIG. 5, the inactivated telomerase could be effectively expressed in the recipient and was specific to the telomere ends in the nucleus. It should be noted that the recipient may be a cardiomyocyte model and a mouse model differentiated from human-derived stem cells, or any other dividing cells. As shown in Figures 6 and 7, telomerase activity test (TRAP Assay) (Figure 6) and chromatin co-immunoprecipitation (ChIP) qPCR (ChIP-qPCR) (Figure 7) demonstrated that the double mutant inactivated telomerase in the technical solution of the present invention is inactive and precisely specific on the deprotected telomere ends.

[0057] Table 8 shows the expression level of CI-TERT measured by qPCR, demonstrating that CI-TERT can be overexpressed in the receptor.

[0058] [Table 8]

[0059] As shown in FIG. 8, in the embodiments of the present invention, it has been demonstrated that adenovirus-inactivated telomerase or artificial-mRNA telomerase can effectively protect telomere ends from shortening, and can suppress a series of cellular reactions caused by abnormal telomere shortening, such as nucleic acid damage, reduction in the number of mitochondria, and reduced contractile force.

[0060] Table 9 shows the measurement of the length of telomere ends injected with inactivated telomerase CI-TERT, which demonstrated that inactivated telomerase CI-TERT effectively protected telomere ends and did not lengthen telomere ends.

[0061] [Table 9]

[0062] Table 10 shows the measurement of nucleic acid damage signals when inactivated telomerase CI-TERT is overexpressed, and it was demonstrated that overexpression of inactivated telomerase CI-TERT can reduce nucleic acid damage signals in cells.

[0063] [Table 10]

[0064] Table 11 shows the measurement of the number of mitochondria when inactivated telomerase CI-TERT is overexpressed, and it is demonstrated that overexpression of inactivated telomerase CI-TERT can effectively reduce mitochondrial death.

[0065] [Table 11]

[0066] Table 12 shows the measurement of the contractile force of cardiomyocytes after overexpression of inactivated telomerase CI-TERT, which proves that overexpression of inactivated telomerase CI-TERT can effectively protect the function of cardiomyocytes.

[0067] [Table 12]

[0068] The above examples show that the adenovirus-inactivated telomerase in the technical solution provided by the present invention can effectively prevent abnormal telomere shortening and nucleic acid damage caused by shortening by protecting telomere ends, and can protect mitochondrial function to protect cellular function, thereby preventing cell death and heart failure caused by disease aggravation.

[0069] In the case of cardiomyopathy, abnormal gradual shortening appears at the telomere ends in non-dividing cells, such as cardiac myocytes. The gradual telomere shortening not only corresponds to the worsening process of the disease observed clinically in the test, but also indicates that the worsening of the disease can be prevented by intervention or treatment at a sufficiently early stage, that is, the continuous shortening of telomeres can be prevented. The test has demonstrated that the phenomenon of abnormal telomere shortening can be simulated in cardiac myocytes differentiated from human-derived stem cells, and that a series of indicators of this phenomenon can be effectively alleviated by inactivated telomerase CI-TERT (Catalytically Inactive TERT, inactivated telomerase). The inventor has found through extensive testing that telomere shortening due to heart failure causes nucleic acid damage, causes a reduction in mitochondrial function and number, and affects myocardial contractile function. By protecting the telomere ends with inactivated telomerase CI-TERT, the worsening of the disease can be suppressed, and nucleic acid damage, a reduction in mitochondrial function and number, and a decline in myocardial function can be prevented.

[0070] The above embodiment of the present invention provides the use of the above adenovirus-inactivated telomerase in the treatment of diseases associated with telomere shortening in non-dividing cells. The therapeutic method of injecting adenovirus-inactivated telomerase can control the expression of telomerase, i.e., the type of recipient cell, with an effective vector, and prevent diseases or symptoms caused by deprotection and shortening of telomere ends in non-dividing cell systems, such as cardiomyocytes. Diseases associated with telomere shortening in cardiomyocytes include, but are not limited to, dilated cardiomyopathy (DCM), hypertrophic cardiomyopathy (HCM), laminopathies, and rare telomere shortening heart diseases (e.g., Duchenne muscular dystrophy (DMD)). The disease samples selected in this embodiment include, but are not limited to, various diseases associated with telomere shortening in cardiomyocytes, such as dilated cardiomyopathy (DCM), hypertrophic cardiomyopathy (HCM), laminopathies, and rare telomere shortening heart diseases (e.g., Duchenne muscular dystrophy (DMD)).

[0071] Of course, the same test effect can be obtained by injecting artificial mRNA-inactivated telomerase mmRNA-CI-TERT instead of adenovirus-inactivated telomerase. After artificial mRNA and adenovirus are injected into the recipient as the vector of the double mutant inactivated telomerase of the present invention, both generate double mutant inactivated telomerase in the recipient, thereby retaining telomeres in the cell nucleus and protecting telomere ends from being detached, and treating and preventing diseases and cell death caused by telomere shortening.

[0072] In view of the above, the technical solution of the present invention uses double-mutated adenovirus-inactivated telomerase and artificial mRNA-inactivated telomerase to retain telomerase in the cell nucleus to protect the telomere end while reducing the risk of canceration, and inhibits the aggravation of diseases caused by telomere shortening.Compared to the prior art, it can prevent telomere shortening, prevent and treat various diseases and aging associated with telomere shortening, inhibit cell death caused by telomere shortening, and extend the telomeres of various diseases and aging cells associated with telomere shortening, and has a certain therapeutic effect on alleviated diseases.

[0073] Of course, the present invention may have other embodiments, and those skilled in the art may make various corresponding modifications and variations based on the present invention without departing from the spirit and essence of the present invention, but all of these corresponding modifications and variations fall within the scope of protection of the claims of the present invention.

Claims

1. An inactivated telomerase, wherein the inactivated telomerase is a double mutant telomerase having double mutation points Y707F and D868A, respectively, and the double mutant telomerase is derived from human-derived telomerase.

2. An adenovirus-inactivated telomerase comprising a double mutant telomerase, the double mutation sites of which are Y707F and D868A, respectively, and the double mutant telomerase is derived from human telomerase.

3. An artificial mRNA-inactivated telomerase comprising a double mutant telomerase, the double mutation sites of which are Y707F and D868A, respectively, and the double mutant telomerase is derived from human telomerase.

4. A therapeutic agent for a disease associated with telomere shortening in non-dividing cells, comprising the adenovirus-inactivated telomerase according to claim 2 or the artificial mRNA-inactivated telomerase according to claim 3.

5. The therapeutic agent according to claim 4, wherein the non-dividing cells include cardiac muscle cells, skeletal muscle cells or neuronal cells.

6. The therapeutic agent according to claim 4, characterized in that the disease associated with telomere shortening in non-dividing cells preferably includes a disease associated with telomere shortening in cardiomyocytes.

7. The therapeutic agent according to claim 6, characterized in that the disease associated with telomere shortening of cardiomyocytes includes dilated cardiomyopathy (DCM), hypertrophic cardiomyopathy (HCM), laminopathies or rare telomere shortening heart diseases.

8. The therapeutic agent according to claim 7, characterized in that the rare telomere shortening heart disease includes Duchenne muscular dystrophy (DMD).

Images